Neisserial vaccine compositions comprising a combination of antigens

ABSTRACT

The present invention relates to immunogenic compositions and vaccines for the treatment and prevention of Neisserial disease. Immunogenic compositions of the invention contain combinations of antigens selected from at least two different classes of antigens including adhesins, autotransporter proteins, toxins, iron acquisitions proteins and membrane-associated protein (preferably integral outer membrane protein)s. Such combinations of antigens are able to target the immune response against different aspects of the neisserial life cycle, resulting in a more effective immune response.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/523,117 filed on Oct. 13, 2005; which was filed pursuant to 35 U.S.C.§371 as a U.S. National Phase Application of International PatentApplication No. PCT/EP03/08571 filed on Jul. 31, 2003; which claimspriority from Great Britain Application Nos. 0305028.3 filed on Mar. 5,2003; GB0230170.3, GB0230168.7, and GB0230164.6 filed on Dec. 24, 2002;GB0225531.3 and GB0225524.8 filed on Nov. 1, 2002; GB0220199.4 andGB0220197.8, filed on Aug. 30, 2002; and GB0218051.1, GB0218035.4,GB0218036.2, and GB0218037.0 filed on Aug. 2, 2002.

TECHNICAL FIELD

The present invention relates to the field of Neisserial immunogeniccompositions and vaccines, their manufacture and the use of suchcompositions in medicine. More particularly, it relates to vaccinecompositions comprising a combination of antigens which have qualitiesallowing the vaccines of the invention to elicit a surprising goodimmune response as measured in a protection assay or a serumbactericidal assay.

BACKGROUND

Neisserial strains of bacteria are the causative agents for a number ofhuman pathologies, against which there is a need for effective vaccinesto be developed. In particular Neisseria gonorrhoeae and Neisseriameningitidis cause pathologies which could be treated by vaccination.

Neisseria gonorrhoeae is the etiologic agent of gonorrhea, one of themost frequently reported sexually transmitted diseases in the world withan estimated annual incidence of 62 million cases (Gerbase et al 1998Lancet 351; (Suppl 3) 2-4). The clinical manifestations of gonorrheainclude inflammation of the mucus membranes of the urogenital tract,throat or rectum and neonatal eye infections. Ascending gonococcalinfections in women can lead to infertility, ectopic pregnancy, chronicpelvic inflammatory disease and tubo-ovarian abscess formation.Septicemia, arthritis, endocarditis and menigitis are associated withcomplicated gonorrhea.

The high number of gonococcal strains with resistance to antibioticscontributes to increased morbidity and complications associated withgonorrhea. An attractive alternative to treatment of gonorrhea withantibiotics would be its prevention using vaccination. No vaccinecurrently exists for N. gonorrhoeae infections.

Neisseria meningitidis is an important pathogen, particularly inchildren and young adults. Septicemia and meningitis are the mostlife-threatening forms of invasive meningococcal disease (IMD). Thisdisease has become a worldwide health problem because of its highmorbidity and mortality.

Thirteen N. meningitidis serogroups have been identified based onantigenic differences in the capsular polysaccharides, the most commonbeing A, B and C which are responsible for 90% of disease worldwide.Serogroup B is the most common cause of meningococcal disease in Europe,USA and several countries in Latin America.

Vaccines based on the capsular polysaccharide of serogroups A, C, W andY have been developed and have been shown to control outbreaks ofmeningococcal disease (Peltola et al 1985 Pediatrics 76; 91-96). Howeverserogroup B is poorly immunogenic and induces only a transient antibodyresponse of a predominantly IgM isotype (Ala'Aldeen D and Cartwright K1996, J. Infect. 33; 153-157). There is therefore no broadly effectivevaccine currently available against the serogroup B meningococcus whichis responsible for the majority of disease in most temperate countries.This is particularly problematic since the incidence of serotype Bdisease is increasing in Europe, Australia and America, mostly inchildren under 5. The development of a vaccine against serogroup Bmeningococcus presents particular difficulties because thepolysaccharide capsule is poorly immunogenic owing to its immunologicsimilarity to human neural cell adhesion molecule. Strategies forvaccine production have therefore concentrated on the surface exposedstructures of the meningococcal outer membrane but have been hampered bythe marked variation in these antigens among strains.

Further developments have led to the introduction of vaccines made up ofouter membrane vesicles which will contain a number of proteins thatmake up the normal content of the bacterial membrane. One of these isthe VA-MENGOC-BC® Cuban vaccine against N. meningitidis serogroups B andC (Rodriguez et al 1999 Mem Inst. Oswaldo Cruz, R10 de Janeiro 94;433-440). This vaccine was designed to combat an invasive meningococcaldisease outbreak in Cuba which had not been eliminated by a vaccinationprogramme using a capsular polysaccharide AC vaccine. The prevailingserogroups were B and C and the VA-MENGOC-BC® vaccine was successful atcontrolling the outbreak with an estimated vaccine efficiency of 83%against serogroup B strains of N. meningitidis (Sierra et al 1990 InNeisseria, Walter Gruyter, Berlin, m. Atchman et al (eds) p 129-134,Sierra et al 1991, NIPH Ann 14; 195-210). This vaccine was effectiveagainst a specific outbreak, however the immune response elicited wouldnot protect against other strains of N. meningitidis.

Subsequent efficacy studies conducted in Latin America during epidemicscaused by homologous and heterologous serogroup B meningococcal strainshave shown some efficacy in older children and adults but itseffectiveness was significantly lower in younger children who are atgreatest risk of infection (Milagres et al 1994, Infect. Immun 62;4419-4424). It is questionable how effective such a vaccine would be incountries with multistrain endemic disease such as the UK. Studies ofimmunogenicity against heterologous strains have demonstrated onlylimited cross-reactive serum bactericidal activity, especially ininfants (Tappero et al 1999, JAMA 281; 1520-1527).

A second outer membrane vesicle vaccine was developed in Norway using aserotype β isolate typical of those prevalent in Scandinavia (Fredriksenet al 1991, NIPH Ann, 14; 67-80). This vaccine was tested in clinicaltrials and found to have a protective efficacy after 29 months of 57%(Bjune et al 1991, Lancet, 338; 1093-1096). However, the use of outermembrane vesicles in vaccines is associated with some problems. Forinstance, the OMV contain toxic lipopolysaccharides and they may containimmunodominant antigens which are either strain specific or areexpressed variably. Several processes have been described which could beused to overcome some of the problems of outer membrane vesiclepreparation vaccines. WO01/09350 describes processes that address someof these problems for instance by reducing toxicity and modifying theantigens present on the outer membrane vesicles.

WO01/52885 describes the possibility of combining outer membranevesicles with other antigens and a list of over 2,000 potentialNeisserial proteins is included from which it is speculated that avaccines with efficacy against a broader range of serotypes could bedeveloped.

There are diverse problems with the anti-meningococcal vaccinescurrently available. The protein based outer membrane vaccines tend tobe specific and effective against only a few strains. The polysaccharidevaccines are also suboptimal since they tend to elicit poor and shortimmune responses, particularly against serogroup B (Lepow et al 1986;Peltola 1998, Pediatrics 76; 91-96).

Neisseria infections represent a considerable health care problem forwhich no vaccines are available in the case of N. gonorrhoeae orvaccines with limitations on their efficacy and ability to protectagainst heterologous strains are available in the case of N.meningitidis. Clearly there is a need to develop superior vaccinesagainst Neisserial infections that will improve on the efficacy ofcurrently available vaccines and allow for protection against a widerrange of strains.

DESCRIPTION OF FIGURES

FIG. 1.—Detection of TbpA and Hsf in OMV's prepared from a recombinantN. meningitidis strain up-regulated for the expression of tbpA and hsf.Separation of OMV preparations (10 μg) by SDS-PAGE analysis (4-20%gradient gels) stained with Coomassie brilliant blue.

FIG. 2.—Detection of recombinant Hsf passenger domain produced in E.coli, bug of purified Hsf passenger protein (Lane 2 & 3) was separatedby SDS-PAGE on a 12% gel in comparison to a molecular weight marker(Lane 1).

FIG. 3.—Analysis of Hap passenger purity as detected by (A) Coomassiestaining, (B) silver staining, (C) anti-His5 western blotting and (D)anti-E. coli. 10 μg of purified antigens was separated by SDS-PAGE on a4-20% acrylamide gradient gel.

FIG. 4.—Regions of sequence similarity shared by FrpA and FrpC proteinsisolated from N. meningitidis strain FAM20. (A) Domain organization ofN. meningitidis strain FAM20 RTX proteins FrpA and FrpC. (B) FrpA/CAmplification products obtained from N. meningitidis strain H44/76.

FIG. 5.—Expression of recombinant Frp23 (FrpA/C conserved region with 23repeats) antigen in E. coli. SDS-PAGE analysis of non-induced (NI) andinduced (I) total cell extracts of E. coli BL21DE3 tranformed withcontrol vectors (pET24d) or recombinant constructs (Frp3, Frp13 and Frp23 respectively). Gels were stained with Coomassie blue (A) ortransferred to nitrocellulose and immuno-detected with anti-His6 mouseserum.

FIG. 6.—Preferred DNA sequence of the FHAB ⅔^(rd) fragment expressed inE. coli.

FIG. 7.—Purification of recombinant FHAB ⅔^(rd) from induced E. coliB121DE3 extracts. (A) Main steps in the purification process. (B)SDS-PAGE analysis of protein fractions sampled at different steps of thepurification process.

FIG. 8.—Adhesion blocking activities of anti-sera directed against theFHAB⅔^(rd), Hap, Hap passenger domain, Hsf and Hsf passenger domainantigens of N. meningitidis.

FIG. 9.—A Coomassie stained gel showing expression levels of Hsf, TbpAand NspA in outer membrane vesicle preparations derived from differentN. meningitidis stains. Lane 1—molecular weight markers; lane 2—outermembrane vesicles prepared from strain H44/76 in which capsularpolysaccharides were downregulated; lane 3—outer membrane vesiclesprepared from strain H44/76 in which capsular polysaccharides and PorAwere downregulated; lane 4—outer membrane vesicles prepared from strainH44/76 in which capsular polysaccharides and PorA were downregulated andNspA was upregulated; lane 5—outer membrane vesicles prepared fromstrain H44/76 in which capsular polysaccharides and PorA weredown-regulated and Hsf was upregulated; lane 6—outer membrane vesiclesprepared from strain H44/76 in which capsular polysaccharides and PorAwere downregulated and TbpA was upregulated; lane 7—outer membranevesicles prepared from strain H44/76 in which capsular polysaccharidesand PorA were downregulated and TbpA and Hsf were upregulated; lane8—outer membrane vesicles prepared from strain H44/76 in which capsularpolysaccharides and PorA were downregulated and TbpA and NspA wereupregulated.

DETAILED DESCRIPTION

The present invention discloses particular combinations of Neisserialantigens which when combined, lead to a surprising enhancement of theefficacy of the vaccine against Neisserial infection.

Neisserial infections progress through several different stages. Forexample, the meningococcal life cycle involve nasopharyngealcolonisation, mucosal attachment, crossing into the bloodstream,multiplication in the blood, induction of toxic shock, crossing theblood/brain barrier and multiplication in the cerebrospinal fluid and/orthe meninges. Different molecules on the surface of the bacterium willbe involved in different steps of the infection cycle. By targeting theimmune response against an effective amount of a combination ofparticular antigens, involved in different processes of Neisserialinfection, a Neisserial vaccine with surprisingly high efficacy can beachieved.

In particular, combinations of certain antigens from different classes,some of which are involved in adhesion to host cells, some of which areinvolved in iron acquisition, some of which are autotransporters andsome of which are toxins, can elicit an immune response which protectsagainst multiple stages of infection. Such combinations of antigens cansurprisingly lead to improved (preferably synergistically improved)vaccine efficacy against Neisserial infection where more that onefunction of the bacterium is targeted by the immune response in anoptimal fashion.

The efficacy of vaccines can be assessed through a variety of assays.Protection assays in animal models are well known in the art.Furthermore, serum bactericidal assay (SBA) is the most commonly agreedimmunological marker to estimate the efficacy of a meningococcal vaccine(Perkins et al. J Infect Dis. 1998, 177:683-691).

Some combinations of antigens (for example, combinations of certainautotransporter proteins and certain iron acquisition proteins) can leadto improved protection in animal model assays and/or synergisticallyhigher SBA titres. Without wishing to be bound by theory, suchsynergistic combinations of antigens are enabled by a number ofcharacteristics of the immune response to the antigen combination. Theantigens themselves are usually surface exposed on the Neisserial cellsand tend to be conserved but also tend not to be present in sufficientquantity on the surface cell for an optimal bactericidal response totake place using antibodies elicited against the antigen alone.Combining the antigens of the invention can result in a formulationeliciting an advantageous combination of bactericidal antibodies whichinteract with the Neisserial cell beyond a critical threshold. At thiscritical level, sufficient antibodies of sufficient quality bind to thesurface of the bacterium to allow efficient killing by complement andmuch higher bactericidal effects are seen as a consequence.

As serum bactericidal assays (SBA) closely reflect the efficacy ofvaccine candidates, the attainment of good SBA titres by a combinationof antigens is a good indication of the protective efficacy of a vaccinecontaining that combination of antigens. The invention relates to theuse of a combination of two antigens either isolated or enriched in amixture with other antigens. When combined, such antigen combinationsinteract advantageously, and preferably synergistically to elicit animmune response that is higher in terms of bactericidal activity (forexample as measured by serum bactericidal assay or SBA), and preferablyhigher than the additive response elicited by the antigens individually,more preferably by a factor of at least 1.2, 1.5, two, three, four,five, six, seven, eight, nine, most preferably by a factor of at leastten.

An additional advantage of the invention is that the combination of theantigens of the invention from different families of proteins in animmunogenic composition may enable protection against a wider range ofstrains.

The invention relates to immunogenic compositions comprising a plurality(two or more) of proteins selected from at least two differentcategories of protein, having different functions within Neisseria.Examples of such categories of proteins are adhesins, autotransporterproteins, toxins, integral outer membrane proteins and Fe acquisitionproteins. The vaccine combinations of the invention show surprisingimprovement in vaccine efficacy against homologous Neisserial strains(strains from which the antigens are derived) and preferably alsoagainst heterologous Neisserial strains.

The invention provides immunogenic compositions comprising at least orexactly two, three, four, five six, seven, eight, nine or ten ofdifferent antigens selected from at least or exactly two, three, four orall five categories of antigens selected from the following:

-   -   at least one Neisserial adhesin;    -   at least one Neisserial autotransporter;    -   at least one Neisserial toxin;    -   at least one Neisserial Fe acquisition protein;    -   at least one Neisserial membrane-associated protein (preferably        outer membrane protein, particularly integral outer membrane        protein).

Preferably, the invention provides immunogenic compositions thatcomprise at least or exactly two, three, four, five, six, seven, eight,nine or ten different Neisseria antigens. Most preferably these antigensare selected from at least or exactly two, three, four or five groups ofproteins selected from the following:

-   -   at least one Neisserial adhesin selected from the group        consisting of FhaB, NspA PilC, Hsf, Hap, MafA, MafB, Omp26, NMB        0315, NMB 0995, NMB 1119 and NadA;    -   at least one Neisserial autotransporter selected from the group        consisting of Hsf, Hap, IgA protease, AspA, and NadA;    -   at least one Neisserial toxin selected from the group consisting        of FrpA, FrpC, FrpA/C, VapD, NM-ADPRT and either or both of LPS        immunotype L2 and LPS immunotype L3;    -   at least one Neisserial Fe acquisition protein selected from the        group consisting of TbpA, TbpB, LbpA, LbpB, HpuA, HpuB, Lipo28        (GNA2132), Sibp, NMB0964, NMB0293, FbpA, Bcp, BfrA, BfrB and        P2086 (XthA); and    -   at least one Neisserial membrane-associated protein, preferably        outer membrane protein, particularly integral outer membrane        protein, selected from the group consisting of PilQ, OMP85,        FhaC, NspA, TbpA, LbpA, TspA, TspB, TdfH, PorB, M1tA, HpuB,        HimD, HisD, GNA1870, OstA, HlpA (GNA1946), NMB 1124, NMB 1162,        NMB 1220, NMB 1313, NMB 1953, HtrA, and PldA (Omp1A).

The antigens of the present invention are all isolated, meaning thatthey are altered by the hand of man. Preferably they are purified tosome degree, most preferably more than 40, 50, 60, 70, 80, 90, 95 or 99%pure (before combination with the other components of the immunogeniccompositions of the invention).

Preferably the immunogenic composition of the invention comprises atleast one Neisserial adhesin and at least one Neisserial autotranporterand optionally a Neisserial toxin, a Neisserial Fe acquisition proteinor a Neisserial membrane-associated protein (preferably integral outermembrane protein). Preferably the antigens are selected from the abovenamed antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial adhesin and at least one Neisserial toxin andoptionally a Neisserial autotranporter, a Neisserial Fe acquisitionprotein or a Neisserial membrane-associated protein (preferably integralouter membrane protein). Preferably the antigens are selected from theabove named antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial adhesin and at least one Neisserial Fe acquisitionprotein and optionally a Neisserial toxin, a Neisserial autotransporteror a Neisserial membrane-associated protein (preferably integral outermembrane protein). Preferably the antigens are selected from the abovenamed antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial adhesin and at least one Neisserialmembrane-associated protein (preferably integral outer membrane protein)and optionally a Neisserial toxin, a Neisserial Fe acquisition proteinor a Neisserial autotransporter. Preferably the antigens are selectedfrom the above named antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial autotranporter and at least one Neisserial toxinand optionally a Neisserial adhesin, a Neisserial Fe acquisition proteinor a Neisserial membrane-associated protein (preferably integral outermembrane protein). Preferably the antigens are selected from the abovenamed antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial autotranporter and at least one Neisserial Feacquisition protein and optionally a Neisserial adhesin, a Neisserialtoxin or a Neisserial membrane-associated protein (preferably integralouter membrane protein). Preferably the antigens are selected from theabove named antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial autotranporter and at least one Neisserialmembrane-associated protein (preferably integral outer membrane protein)and optionally a Neisserial adhesin, a Neisserial Fe acquisition proteinor a Neisserial toxin. Preferably the antigens are selected from theabove named antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial toxin and at least one Neisserial Fe acquisitionprotein and optionally a Neisserial adhesin, a Neisserialautotransporter or a Neisserial membrane-associated protein (preferablyintegral outer membrane protein). Preferably the antigens are selectedfrom the above named antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial toxin and at least one Neisserialmembrane-associated protein (preferably integral outer membrane protein)and optionally a Neisserial adhesin, a Neisserial autotransporter or aNeisserial toxin. Preferably the antigens are selected from the abovenamed antigens.

Preferably the immunogenic composition of the invention comprises atleast one Neisserial Fe acquisition protein and at least one Neisserialmembrane-associated protein (preferably integral outer membrane protein)and optionally a Neisserial adhesin, a Neisserial autotransporter or aNeisserial toxin. Preferably the antigens are selected from the abovenamed antigens.

Preferably all five groups of antigen are represented in the immunogeniccomposition of the invention.

Where a protein is specifically mentioned herein, it is preferably areference to a native, full-length protein, and to its natural variants(i.e. to a native protein obtainable from a Neisserial, preferablymeningococcal strain) but it may also encompass antigenic fragmentsthereof (particularly in the context of subunit vaccines). These arefragments (often specifically described herein) containing or comprisingat least 10 amino acids, preferably 20 amino acids, more preferably 30amino acids, more preferably 40 amino acids or most preferably 50 aminoacids, taken contiguously from the amino acid sequence of the protein.In addition, antigenic fragments denotes fragments that areimmunologically reactive with antibodies generated against theNeisserial full-length proteins or with antibodies generated byinfection of a mammalian host with Neisseria. Antigenic fragments alsoincludes fragments that when administered at an effective dose, elicit aprotective immune response against Neisserial infection, more preferablyit is protective against N. meningitidis and/or N. gonorrhoeaeinfection, most preferably it is protective against N. meningitidisserogroup B infection.

Also included in the invention are recombinant fusion proteins ofNeisserial proteins of the invention, or fragments thereof. These maycombine different Neisserial proteins or fragments thereof in the samepolypeptide. Alternatively, the invention also includes individualfusion proteins of Neisserial proteins or fragments thereof, as a fusionprotein with heterologous sequences such as a provider of T-cellepitopes or purification tags, for example: β-galactosidase,glutathione-S-transferase, green fluorescent proteins (GFP), epitopetags such as FLAG, myc tag, poly histidine, or viral surface proteinssuch as influenza virus haemagglutinin, tetanus toxoid, diphtheriatoxoid, CRM197.

Antigens of the Invention

NMB references refer to reference numbers to sequences which can beaccessed from www.neisseria.org.

1. Adhesins

Adhesins include FhaB (WO98/02547), NadA (J. Exp.Med (2002) 195:1445;NMB 1994), Hsf also known as NhhA (NMB 0992) (WO99/31132), Hap (NMB1985)(WO99/55873), NspA (WO96/29412), MafA (NMB 0652) and MafB (NMB0643) (Annu Rev Cell Dev Biol. 16; 423-457 (2000); Nature Biotech 20;914-921 (2002)), Omp26 (NMB 0181), NMB 0315, NMB 0995, NMB 1119 and PilC(Mol. Microbio1.1997, 23; 879-892). These are proteins that are involvedin the binding of Neisseria to the surface of host cells. Hsf is anexample of an adhesin, as well as being an autotranporter proteinImmunogenic compositions of the invention may therefore includecombinations of Hsf and other autotransporter proteins where Hsfcontributes in its capacity as an adhesin. These adhesins may be derivedfrom Neisseria meningitidis or Neisseria gonorrhoeae or other Neisserialstrains. The invention also includes other adhesins from Neisseria.

FhaB

This antigen has been described in WO98/02547 SEQ ID NO 38 (nucleotides3083-9025)—see also NMB0497. The present inventors have found FhaB to beparticularly effectively at inducing anti-adhesive antibodies alone andin particular with other antigens of the invention. Although full lengthFhaB could be used, the inventors have found that particular C-terminaltruncates are surprisingly at least as effective and preferably evenmore effective in terms of cross-strain effect. Such truncates have alsobeen advantageously shown to be far easier to clone. FhaB truncates ofthe invention typically correspond to the N-terminal two-thirds of theFhaB molecule, preferably the new C-terminus being situated at position1200-1600, more preferably at position 1300-1500, and most preferably atposition 1430-1440. Specific embodiments have the C-terminus at 1433 or1436. Accordingly such FhaB truncates of the invention and vaccinescomprising such truncates are independent aspects of the presentinvention as well as being components of the combination immunogeniccompositions of the invention. The N-terminus may also be truncated byup to 10, 20, 30, 40 or 50 amino acids.

2. Autotransporter Proteins

Autotransporter proteins typically are made up of a signal sequence, apassenger domain and an anchoring domain for attachment to the outermembrane. Examples of autotransporter proteins include Hsf (WO99/31132)(NMB 0992), HMW, Hia (van Ulsen et al Immunol. Med. Microbiol. 2001 32;53-64), Hap (NMB 1985) (WO99/55873; van Ulsen et al Immunol. Med.Microbiol. 2001 32; 53-64), UspA, UspA2, NadA (NMB 1994) (Comanducci etal J. Exp. Med. 2002 195; 1445-1454), AspA (Infection and Immunity 2002,70(8); 4447-4461; NMB 1029), Aida-1 like protein, SSh-2 and Tsh. NadA(J. Exp.Med (2002) 195:1445) is another example of an autotransporterproteins, as well as being an adhesin. Immunogenic compositions of theinvention may therefore include combinations of NadA and adhesins whereNadA contributes in its capacity as an autotransporter protein.Theseproteins may be derived from Neisseria meningitidis or Neisseriagonorrhoeae or other Neiserial strians. The invention also includesother autotransporter proteins from Neisseria.

Hsf

Hsf has a structure that is common to autotransporter proteins. Forexample, Hsf from N. meningitidis strain H44/76 consists of a signalsequence made up of amino acids 1-51, a head region at the aminoterminus of the mature protein (amino acids 52-479) that is surfaceexposed and contains variable regions (amino acids 52-106, 121-124,191-210 and 230-234), a neck region (amino acids 480-509), a hydrophobicalpha-helix region (amino acids 518-529) and an anchoring domain inwhich four transmembrane strands span the outer membrane (amino acids539-591).

Although full length Hsf may be used in immunogenic compositions of theinvention, various Hsf truncates and deletions may also beadvantageously used depending on the type of vaccine.

Where Hsf is used in a subunit vaccine, it is preferred that a portionof the soluble passenger domain is used; for instance the completedomain of amino acids 52 to 479, most preferably a conserved portionthereof, for instance the particularly advantageous sequence of aminoacids 134 to 479. Preferred forms of Hsf may be truncated so as todelete variable regions of the protein disclosed in WO01/55182.Preferred variants would include the deletion of one, two, three, four,or five variable regions as defined in WO01/55182. The above sequencesand those described below, can be extended or truncated by up to 1, 3,5, 7, 10 or 15 amino acids at either or both N or C termini.

Preferred fragments of Hsf therefore include the entire head region ofHsf, preferably containing amino acids 52-473. Additional preferredfragments of Hsf include surface exposed regions of the head includingone or more of the following amino acid sequences; 52-62, 76-93,116-134, 147-157, 157-175, 199-211, 230-252, 252-270, 284-306, 328-338,362-391, 408-418, 430-440 and 469-479.

Where Hsf is present in an outer membrane vesicle preparation, it may beexpressed as the full-length protein or preferably as an advantageousvariant made up of a fusion of amino acids 1-51 and 134-591 (yielding amature outer membrane protein of amino acid sequence 134 to theC-terminus). Preferred forms of Hsf may be truncated so as to deletevariable regions of the protein disclosed in WO01/55182. Preferredvariants would include the deletion of one, two, three, four, or fivevariable regions as defined in WO01/55182. Preferably the first andsecond variable regions are deleted. Preferred variants would deleteresidues from between amino acid sequence 52 through to 237 or 54through to 237, more preferably deleting residues between amino acid 52through to 133 or 55 through to 133. The mature protein would lack thesignal peptide.

Hap

Computer analysis of the Hap-like protein from Neisseria meningitidisreveals at least three structural domains. Considering the Hap-likesequence from strain H44/76 as a reference, Domain 1, comprisingamino-acid 1 to 42, encodes a sec-dependant signal peptidecharacteristic of the auto-transporter family, Domain 2, comprisingamino-acids 43 to 950, encode the passenger domain likely to be surfaceexposed and accessible to the immune system, Domain 3, comprisingresidues 951 to the C-terminus (1457), is predicted to encode abeta-strands likely to assemble into a barrel-like structure and to beanchored into the outer-membrane. Since domains 2 is likely to besurface-exposed, well conserved (more than 80% in all strain tested) andcould be produced as subunit antigens in E. coli, it represents aninteresting vaccine candidates. Since domains 2 and 3 are likely to besurface-exposed, are well conserved (Pizza et al. (2000), Science 287:1816-1820), they represent interesting vaccine candidates. Domain 2 isknown as the passenger domain.

Immunogenic compositions of the invention may comprise the full-lengthHap protein, preferably incorporated into an OMV preparation.Immunogenic compositions of the invention may also comprise thepassenger domain of Hap which in strain H44/76 is composed of amino acidresidues 43-950. This fragment of Hap would be particularlyadvantageously used in a subunit composition of the invention. The abovesequence for the passenger domain of Hap can be extended or truncated byup to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids at either or both Nor C termini.

3. Iron Acquisition Proteins

Iron aquisition proteins include TbpA (NMB 0461) (WO92/03467, U.S. Pat.No. 5,912,336, WO93/06861 and EP586266), TbpB (NMB 0460) (WO93/06861 andEP586266), LbpA (NMB 1540) (Med Microbiol (1999) 32:1117), LbpB (NMB1541)(WO/99/09176), HpuA (U73112.2) (Mol. Microbiol. 1997, 23; 737-749),HpuB (NC_(—)003116.1) (Mol. Microbiol. 1997, 23; 737-749), P2086 alsoknown as XthA (NMB 0399) (13^(th) International Pathogenic NeisseriaConference 2002), FbpA (NMB 0634), FbpB, BfrA (NMB 1207), BfrB (NMB1206), Lipo28 also known as GNA2132 (NMB 2132), Sibp (NMB 1882), HmbR,HemH, Bcp (NMB 0750), Iron (III) ABC transporter-permease protein(Tettelin et al Science 287; 1809-1815 2000), Iron (III) ABCtransporter—periplasmic (Tettelin et al Science 287; 1809-1815 2000),TonB-dependent receptor (NMB 0964 and NMB 0293)(Tettelin et al Science287; 1809-1815 2000) and transferrin binding protein related protein(Tettelin et al Science 287; 1809-1815 2000). These proteins may bederived from Neisseria meningitidis, Neisseria gonorrhoeae or otherNeisserial strains. The invention also includes other iron acquisitionproteins from Neisseria.

TbpA

TbpA interacts with TbpB to form a protein complex on the outer membraneof Neisseria, which binds transferrin. Structurally, TbpA contains anintracellular N-terminal domain with a TonB box and plug domain,multiple transmembrane beta strands linked by short intracellular andlonger extracellular loops.

Two families of TbpB have been distinguished, having a high molecularweight and a low molecular weight respectively. High and low molecularweight forms of TbpB associate with different families of TbpA which aredistinguishable on the basis of homology. Despite being of similarmolecular weight, they are known as the high molecular weight and lowmolecular weight families because of their association with the high orlow molecular weight form of TbpB (Rokbi et al FEMS Microbiol. Lett.100; 51, 1993). The terms TbpA(high) and TbpA(low) are used to refer tothese two forms of TbpA, and similarly for TbpB Immunogenic compositionsof the invention may comprise TbpA and TbpB from serogroups A, B, C, Yand W-135 of N. meningitidis as well as iron acquisition proteins fromother bacteria including N. gonorrhoeae. Transferrin binding proteinsTbpA and TbpB have also been referred to as Tbp1 and Tbp2 respectively(Cornelissen et al Infection and Immunity 65; 822, 1997).

TbpA contains several distinct regions. For example, in the case of TbpAfrom N. meningitidis strain H44/76, the amino terminal 186 amino acidsform an internal globular domain, 22 beta strands span the membrane,forming a beta barrel structure. These are linked by short intracellularloops and larger extracellular loops.

Extracellular loops 2, 3 and 5 have the highest degree of sequencevariability and loop 5 is surface exposed. Loops 5 and 4 are involved inligand binding.

Preferred fragments of TbpA include the extracellular loops of TbpA.Using the sequence of TbpA from N. meningitidis strain H44/76, theseloops correspond to amino acids 200-202 for loopl, amino acids 226-303for loop 2, amino acids 348-395 for loop 3, amino acids 438-471 for loop4, amino acids 512-576 for loop 5, amino acids 609-625 for loop 6, aminoacids 661-671 for loop 7, amino acids 707-723 for loop 8, amino acids769-790 for loop 9, amino acids 814-844 for loop 10 and amino acids872-903 for loop 11. The corresponding sequences, after sequencealignment, in other Tbp proteins would also constitute preferredfragments. Most preferred fragments would include amino acid sequencesconstituting loop 2, loop 3, loop 4 or loop 5 of Tbp.

Where the immunogenic compositions of the invention comprise TbpA, it ispreferable to include both TbpA(high) and TbpA (low).

Although TbpA is preferably presented in an OMV vaccine, it may also bepart of a subunit vaccine. For instance, isolated iron acquisitionproteins which could be introduced into an immunogenic composition ofthe invention are well known in the art (WO00/25811). They may beexpressed in a bacterial host, extracted using detergent (for instance2% Elugent) and purified by affinity chromatography or using standardcolumn chromatography techniques well known to the art (Oakhill et alBiochem J. 2002 364; 613-6).

Where TbpA is presented in an OMV vaccine, its expression can beupregulated by genetic techiques discussed herein, or may preferably beupregulated by growth of the parent strain under iron limitationconditions as described below. This process will also result in theupregulation of variable iron-regulated proteins, particularly FrpBwhich may become immunodominant and it is therefore advantageous todownregulate the expression of (and preferably delete the genesencoding) such proteins (particularly FrpB) as described below, toensure that the immunogenic composition of the invention elicits animmune response against antigens present in a wide range of Neisserialstrains. It is preferred to have both TbpA(high) and TbpA(low) presentin the immunogenic composition and this is preferably achieved bycombining OMVs derived from two strains, expressing the alternativeforms of TbpA.

4. Toxins

Toxins include FrpA (NMB 0585; NMB 1405), FrpA/C (see below fordefinition), FrpC(NMB 1415; NMB 1405) (WO92/01460), NM-ADPRT (NMB 1343)(13^(th) International Pathogenic Neisseria Conference 2002 Masignani etal p135), VapD (NMB 1753), lipopolysaccharide (LPS; also calledlipooligosaccharide or LOS) immunotype L2 and LPS immunotype L3. FrpAand FrpC contain a region which is conserved between these two proteinsand a preferred fragment of the proteins would be a polypeptidecontaining this conserved fragment, preferably comprising amino acids227-1004 of the sequence of FrpA/C. These antigens may be derived fromNeisseria meningitidis or Neisseria gonorrhoeae or other Neisserialstrains. The invention also includes other toxins from Neisseria.

In an alternative embodiment, toxins may include antigens involved inthe regulation of toxicity, for example OstA which functions in thesynthesis of lipopolysaccharides.

FrpA and FrpC

Neisseria meningitidis encodes two RTX proteins, referred to as FrpA &FrpC secreted upon iron limitation (Thompson et al., (1993) J.Bacteriol. 175:811-818; Thompson et al., (1993) Infect. Immun61:2906-2911). The RTX (Repeat ToXin) protein family have in common aseries of 9 amino acid repeat near their C-termini with the consensus:Leu Xaa Gly Gly Xaa Gly (Asn/Asp) Asp Xaa. (LXGGXGN_(/D)DX). The repeatsin E. coli HlyA are thought to be the site of Ca2+ binding. Asrepresented in FIG. 4, meningococcal FrpA and FrpC proteins, ascharacterized in strain FAM20, share extensive amino-acid similarity intheir central and C-terminal regions but very limited similarity (ifany) at the N-terminus Moreover, the region conserved between FrpA andFrpC exhibit some polymorphism due to repetition (13 times in FrpA and43 times in FrpC) of a 9 amino acid motif Immunogenic compositions ofthe invention may comprise the full length FrpA and/or FrpC orpreferably, a fragment comprising the sequence conserved between FrpAand FrpC. The conserved sequence is made up of repeat units of 9 aminoacids. Immunogenic compositions of the invention would preferablycomprise more that three repeats, more than 10 repeats, more than 13repeats, more than 20 repeats or more than 23 repeats.

Such truncates have advantageous properties over the full lengthmolecules and vaccines comprising such antigens form an independentaspect of invention as sell as being incorporated in the immunogeniccompositions of the invention.

Sequences conserved between FrpA and FrpC are designated FrpA/C andwhereever FrpA or FrpC forms a constituent of immunogenic compositionsof the invention, FrpA/C could be advantageously used Amino acids277-1004 of the FrpA sequence is the preferred conserved region. Theabove sequence can be extended or truncated by up to 1, 3, 5, 7, 10, 15,20, 25, or 30 amino acids at either or both N or C termini.

LPS

LPS (lipopolysaccharide, also known as LOS—lipooligosaccharide) is theendotoxin on the outer membrane of Neisseria. The polysaccharide moietyof the LPS is known to induce bactericidal antibodies.

Heterogeneity within the oligosaccharide moiety of the LPS generatesstructural and antigenic diversity among different neisserial strains(Griffiss et al. Inf. Immun. 1987; 55: 1792-1800). This has been used tosubdivide meningococcal strains into 12 immunotypes (Scholtan et al. JMed Microbiol 1994, 41:236-243) Immunotypes L3, L7, & L9 areimmunologically identical and are structurally similar (or even thesame) and have therefore been designated L3, 7, 9 (or, for the purposesof this specification, generically as “L3”). Meningococcal LPS L3, 7, 9(L3), L2 and L5 can be modified by sialylation, or by the addition ofcytidine 5′-monophosphate-N-acetylneuraminic acid. Although L2, L4 andL6 LPS are distinguishable immunologically, they are structurallysimilar and where L2 is mentioned herein, either L4 or L6 may beoptionally substituted within the scope of the invention. See M. P.Jennings et al, Microbiology 1999, 145, 3013-3021 and Mol Microbiol2002, 43:931-43 for further illustration of LPS structure andheterogeneity.

Where LPS, preferably meningococcal LPS, is included in a vaccine of theinvention, preferably and advantageously either or both of immunotypesL2 and L3 are present. LPS is preferably presented in an outer membranevesicle (preferably where the vesicle is extracted with a low percentagedetergent, more preferably 0-0.5%, 0.02-0.4%, 0.04-0.3%, 0.06-0.2%,0.08-0.15% or 0.1%, most preferably deoxycholate [DOC]) but may also bepart of a subunit vaccine. LPS may be isolated using well in knownprecedure including the hot water-phenol procedure (Wesphal and JannMeth. Carbo. Chem. 5; 83-91 1965). See also Galanos et al. 1969, Eur JBiochem 9:245-249, and Wu et al. 1987, Anal Bio Chem 160:281-289. LPSmay be used plain or conjugated to a source of T-cell epitopes such astetanus toxoid, Diphtheria toxoid, CRM-197 or OMV outer membraneproteins. Techniques for conjugating isolated LOS are also known (seefor instance EP 941738 incorporated by reference herein).

Where LOS (in particular the LOS of the invention) is present in a blebformulation the LOS is preferably conjugated in situ by methods allowingthe conjugation of LOS to one or more outer membrane proteins alsopresent on the bleb preparation (e.g. PorA or PorB in meningococcus).

This process can advantageously enhance the stability and/orimmunogenicity (providing T-cell help) and/or antigenicity of the LOSantigen within the bleb formulation—thus giving T-cell help for theT-independent oligosaccharide immunogen in its most protectiveconformation—as LOS in its natural environment on the surface ofmeningococcal outer membrane. In addition, conjugation of the LOS withinthe bleb can result in a detoxification of the LOS (the Lipid A portionbeing stably buried in the outer membrane thus being less available tocause toxcity). Thus the detoxification methods mentioned herein ofisolating blebs from htrB⁻ or msbB⁻ mutants, or by adding non toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] to the composition (see WO 93/14115, WO 95/03327,Velucchi et al (1997) J Endotoxin Res 4: 1-12, and EP 976402 for furtherdetails of non-toxic peptide functional equivalents of polymyxinB—particularly the use of the peptide SAEP 2 (of sequence KTKCKFLKKCwhere the 2 cysteines form a disulphide bridge)) may not be required(but which may be added in combination for additional security). Thusthe inventors have found that a composition comprising blebs wherein LOSpresent in the blebs has been conjugated in an intra-bleb fashion toouter membrane proteins also present in the bleb can form the basis of avaccine for the treatment or prevention of diseases caused by theorganism from which the blebs have been derived, wherein such vaccine issubstantially non-toxic and is capable of inducing a T-dependentbactericidal response against LOS in its native environment.

This invention therefore further provides such an intra-bleb LOSconjugated meningococcal bleb preparation.

Such bleb preparations may be isolated from the bacterial in question(see WO 01/09350), and then subjected to known conjugation chemistriesto link groups (e.g. NH₂ or COOH) on the oligosaccharide portion of LOSto groups (e.g. NH₂ or COOH) on bleb outer membrane proteins.Cross-linking techniques using glutaraldehyde, formaldehyde, orglutaraldehyde/formaldehyde mixes may be used, but it is preferred thatmore selective chemistries are used such as EDAC or EDAC/NHS (J. V.Staros, R. W. Wright and D. M. Swingle. Enhancement byN-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176). Other conjugationchemistries or treatments capable of creating covalent links between LOSand protein molecules that could be used are described in EP 941738.

Preferably the bleb preparations are conjugated in the absence ofcapsular polysaccharide. The blebs may be isolated from a strain whichdoes not produce capsular polysaccharide (naturally or via mutation asdescribed below), or may be purified from most and preferably allcontaminating capsular polysaccharide. In this way, the intra-bleb LOSconjugation reaction is much more efficient.

Preferably more than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of theLOS present in the blebs is cross-linked/conjugated.

Intrableb conjugation should preferably incorporate 1, 2 or all 3 of thefollowing process steps: conjugation pH should be greater than pH 7.0,preferably greater than or equal to pH 7.5 (most preferably under pH 9);conditions of 1-5% preferably 2-4% most preferably around 3% sucroseshould be maintained during the reaction; NaCl should be minimised inthe conjugation reaction, preferably under 0.1M, 0.05M, 0.01M, 0.005M,0.001M, and most preferably not present at all. All these processfeatures make sure that the blebs remain stable and in solutionthroughout the conjugation process.

The EDAC/NHS conjugation process is a preferred process for intra-blebconjugation. EDAC/NHS is preferred to formalydehyde which can cross-linkto too high an extent thus adversely affecting filterability. EDACreacts with carboxylic acids (such as KDO in LOS) to create anactive-ester intermediate. In the presence of an amine nucleophile (suchas lysines in outer membrane proteins such as PorB), an amide bond isformed with release of an isourea by-product. However, the efficiency ofan EDAC-mediated reaction may be increased through the formation of aSulfo-NHS ester intermediate. The Sulfo-NHS ester survives in aqueoussolution longer than the active ester formed from the reaction of EDACalone with a carboxylate. Thus, higher yields of amide bond formationmay be realized using this two-stage process. EDAC/NHS conjugation isdiscussed in J.V. Staros, R.W. Wright and D. M. Swingle. Enhancement byN-hydroxysuccinimide of water-soluble carbodiimide-mediated couplingreactions. Analytical chemistry 156: 220-222 (1986); and BioconjugatesTechniques. Greg T. Hermanson (1996) pp 173-176. Preferably 0.01-5 mgEDAC/mg bleb is used in the reaction, more preferably 0.05-1 mg EDAC/mgbleb. The amount of EDAC used depends on the amont of LOS present in thesample which in turn depends on the deoxycholate (DOC) % used to extractthe blebs. At low % DOC (e.g. 0.1%), high amounts of EDAC are used (1mg/mg and beyond), however at higher % DOC (e.g. 0.5%), lower amounts ofEDAC are used (0.025-0.1 mg/mg) to avoid too much inter-blebcrosslinking.

A preferred process of the invention is therefore a process forproducing intra-bleb conjugated LOS (preferably meningococcal)comprising the steps of conjugating blebs in the presence of EDAC/NHS ata pH between pH 7.0 and pH 9.0 (preferably around pH 7.5), in 1-5%(preferably around 3%) sucrose, and optionally in conditionssubstantially devoid of NaCl (as described above), and isolating theconjugated blebs from the reaction mix.

The reaction may be followed on Western separation gels of the reactionmixture using anti-LOS (e.g. anti-L2 or anti-L3) mAbs to show theincrease of LOS molecular weight for a greater proportion of the LOS inthe blebs as reaction time goes on.

Yields of 99% blebs can be recovered using such techniques.

EDAC was found to be an excellent intra-bleb cross-linking agent in thatit cross-linked LOS to OMP sufficiently for improved LOS T-dependentimmunogenicity, but did not cross link it to such a high degree thatproblems such as poor filterability, aggregation and inter-blebcross-linking occurred. The morphology of the blebs generated is similarto that of unconjugated blebs (by electron microscope). In addition, theabove protocol avoided an overly high cross-linking to take place (whichcan decrease the immunogenicity of protective OMPs naturally present onthe surface of the bleb e.g. TbpA or Hsf).

It is preferred that the meningococcal strain from which the blebs arederived is a mutant strain that cannot produce capsular polysaccharide(e.g. one of the mutant strains described below, in particular siaff).It is also preferred that immunogenic compositions effective againstmeningococcal disease comprise both an L2 and L3 bleb, wherein the L2and L3 LOS are both conjugated to bleb outer membrane proteins.Furthermore, it is preferred that the LOS structure within theintra-bleb conjugated bleb is consistent with it having been derivedfrom an lgtB⁻ meningococcal strain (as described below). Most preferablyimmunogenic compositions comprise intrableb-conjugated blebs: derivedfrom a mutant meningococcal strain that cannot produce capsularpolysaccharide and is lgtB⁻; comprising L2 and L3 blebs derived frommutant meningococcal strains that cannot produce capsularpolysaccharide; comprising L2 and L3 blebs derived from mutantmeningococcal strains that are lgtB⁻; or most preferably comprising L2and L3 blebs derived from mutant meningococcal strains that cannotproduce capsular polysaccharide and are lgtB⁻.

Typical L3 meningococcal strain that can be used for the presentinvention is H44/76 menB strain. A typical L2 strain is the B16B6 menBstrain or the 39E meningococcus type C strain.

As stated above, the blebs of the invention have been detoxified to adegree by the act of conjugation, and need not be detoxified anyfurther, however further detoxification methods may be used foradditional security, for instance using blebs derived from ameningococcal strain that is htrB⁻ or msbB⁻ or adding a non-toxicpeptide functional equivalent of polymyxin B [a molecule with highaffinity to Lipid A] (preferably SEAP 2) to the bleb composition (asdescribed above).

In the above way meningococcal blebs and immunogenic compositionscomprising blebs are provided which have as an important antigen LOSwhich is substantially non-toxic, devoid of autoimmunity problems, has aT-dependent character, is present in its natural environment, and iscapable of inducing a bactericidal antibody response against more than90% of meningococcal strains (in the case of L2+L3 compositions).

Preferably intrableb LOS conjugation should incorporate 1, 2 or all 3 ofthe following process steps: conjugation pH should be greater than pH7.0, preferably greater than or equal to pH 7.5 (most preferably underpH 9); conditions of 1-5% preferably 2-4% most preferably around 3%sucrose should be maintained during the reaction; NaCl should beminimised in the conjugation reaction, preferably under 0.1M, 0.05M,0.01M, 0.005M, 0.001M, and most preferably not present at all. All theseprocess features make sure that the blebs remain stable and in solutionthroughout the conjugation process.

Although LOS can be conjugated within blebs to outer membrane proteinsby various techniques and chemistries, the EDAC/NHS conjugation processis a preferred process for intra-bleb conjugation. EDAC/NHS is preferredto formalydehyde which can cross-link to too high an extent thusadversely affecting filterability. EDAC reacts with carboxylic acids tocreate an active-ester intermediate. In the presence of an aminenucleophile, an amide bond is formed with release of an isoureaby-product. However, the efficiency of an EDAC-mediated reaction may beincreased through the formation of a Sulfo-NHS ester intermediate. TheSulfo-NHS ester survives in aqueous solution longer than the activeester formed from the reaction of EDAC alone with a carboxylate. Thus,higher yields of amide bond formation may be realized using thistwo-stage process. EDAC/NHS conjugation is discussed in J. V. Staros, R.W. Wright and D. M. Swingle. Enhancement by N-hydroxysuccinimide ofwater-soluble carbodiimide-mediated coupling reactions. Analyticalchemistry 156: 220-222 (1986); and Bioconjugates Techniques. Greg T.Hermanson (1996) pp 173-176.

A preferred process of the invention is therefore a process forproducing intra-bleb conjugated LOS (preferably meningococcal)comprising the steps of conjugating blebs in the presence of EDAC/NHS ata pH between pH 7.0 and pH 9.0 (preferably around pH 7.5), in 1-5%(preferably around 3%) sucrose, and optionally in conditionssubstantially devoid of NaCl (as described above), and isolating theconjugated blebs from the reaction mix.

The reaction may be followed on separation gels of the reaction mixtureusing anti-LOS (e.g. anti-L2 or anti-L3) mAbs to show the increase ofLOS molecular weight for a greater proportion of the LOS in the blebs asreaction time goes on.

Yields of 99% blebs can be recovered using such techniques. EDAC wasfound to be an excellent intra-bleb cross-linking agent in that itcross-linked LOS to OMP sufficiently for improved LOS T-dependentimmunogenicity, but did not cross link it to such a high degree thatproblems such as poor filterability and inter-bleb cross-linkingoccurred. A too high cross-linking should also avoided to avoid anydecrease in immunogenicity of protective OMPs naturally present on thesurface of the bleb e.g. TbpA.

5. Integral Outer Membrane Proteins

Other categories of Neisserial proteins may also be candidates forinclusion in the Neisserial vaccines of the invention and may be able tocombine with other antigens in a surprisingly effective manner. Membraneassociated proteins, particularly integral membrane proteins and mostadvantageously outer membrane proteins, especially integral outermembrane proteins may be used in the compositions of the presentinvention. An example of such a protein is PldA also known as Omp1A (NMB0464) (WO00/15801) which is a Neisserial phospholipase outer membraneprotein. Further examples are TspA (NMB 0341) (Infect. Immun 1999, 67;3533-3541) and TspB (T-cell stimulating protein) (WO 00/03003; NMB 1548,NMB 1628 or NMB 1747). Further examples include PilQ (NMB 1812)(WO99/61620), OMP85—also known as D15—(NMB 0182) (WO00/23593), NspA(U52066) (WO96/29412), FhaC(NMB 0496 or NMB 1780), PorB (NMB 2039) (Mol.Biol. Evol. 12; 363-370, 1995), HpuB (NC_(—)003116.1), TdfH(NMB 1497)(Microbiology 2001, 147; 1277-1290), OstA (NMB 0280), M1tA also known asGNA33 and Lipo30 (NMB0033), HtrA (NMB 0532; WO 99/55872), HimD (NMB1302), HisD (NMB 1581), GNA 1870 (NMB 1870), HlpA (NMB 1946), NMB 1124,NMB 1162, NMB 1220, NMB 1313, NMB 1953, HtrA, TbpA (NMB 0461)(WO92/03467) (see also above under iron acquisition proteins) and LbpA(NMB 1541).

OMP85

Immunogenic compositions of the invention may comprise the full lengthOMP85, preferably as part of an OMV preparation. Fragments of OMP85 mayalso be used in immunogenic compositions of the invention, inparticularly, the surface exposed domain of OMP85 made up of amino acidresidues 1-475 or 50-475 is preferably incorporated into a subunitcomponent of the immunogenic compositions of the invention. The abovesequence for the surface exposed domain of OMP85 can be extended ortruncated by up to 1, 3, 5, 7, 10, 15, 20, 25, or 30 amino acids ateither or both N or C termini. It is preferred that the signal sequenceis omitted from the OMP85 fragment.

OstA

OstA functions in the synthesis of lipopolysaccharides and may beconsidered to be a regulator of toxicity. OstA may alternatively beincluded in the toxin category where the toxin category is broadened tocontain regulators of toxicity as well as toxins.

Immunogenic Compositions

An immunogenic composition is a composition comprising at least oneantigen which is capable of generating an immune response whenadministered to a host. Preferably, such immunogenic preparations arecapable of generating a protective immune response against Neisserial,preferably Neisseria meningitidis or Neisseria gonorrhoeae infection.

The invention relates to immunogenic compositions comprising at leasttwo antigens, which preferably elicit one or more of a synergisticbactericidal, protective, or adhesion blocking response.

SBA Bactericidal Assays of the Invention

Such a synergistic response may be characterised by the SBA elicited bythe combination of antigens being at least 50%, two times, three times,preferably four times, five times, six times, seven times, eight times,nine times and most preferably ten times higher than the SBA elicited byeach antigen separately. Preferably SBA is measured against a homologousstrain from which the antigens are derived and preferably also against apanel of heterologous strains. (See below for a representative panel forinstance BZ10 (B:2b:P1.2) belonging to the A-4 cluster; B16B6(B:2a:P1.2) belonging to the ET-37 complex; and H44/76 (B:15:P1.7, 16)).SBA is the most commonly agreed immunological marker to estimate theefficacy of a meningococcal vaccine (Perkins et al. J Infect Dis. 1998,177:683-691). Satisfactory SBA can be acertained by any known method.SBA can be carried out using sera obtained from animal models (seeexamples 17-20), or from human subjects.

A preferred method of conducting SBA with human sera is the following. Ablood sample is taken prior to the first vaccination, two months afterthe second vaccination and one month after the third vaccination (threevaccinations in one year being a typical human primary vaccinationschedule administered at, for instance, 0, 2 and 4 months, or 0, 1 and 6months). Such human primary vaccination schedules can be carried out oninfants under 1 year old (for instance at the same time as Hibvaccinations are carried out) or 2-4 year olds or adolescents may alsobe vaccinated to test SBA with such a primary vaccination schedule. Afurther blood sample may be taken 6 to 12 months after primaryvaccination and one month after a booster dose, if applicable.

SBA will be satisfactory for an antigen or bleb preparation withhomologous bactericidal activity if one month after the third vaccinedose (of the primary vaccination schedule) (in 2-4 year olds oradolescents, but preferably in infants in the first year of life) thepercentage of subjects with a four-fold increase in terms of SBA(antibody dilution) titre (compared with pre-vaccination titre) againstthe strain of meningococcus from which the antigens of the inventionwere derived is greater than 30%, preferably greater than 40%, morepreferably greater than 50%, and most preferably greater than 60% of thesubjects.

Of course an antigen or bleb preparation with heterologous bactericidalactivity can also constitute bleb preparation with homologousbactericidal activity if it can also elicit satisfactory SBA against themeningococcal strain from which it is derived.

SBA will be satisfactory for an antigen or bleb preparation withheterologous bactericidal activity if one month after the third vaccinedose (of the primary vaccination schedule) (in 2-4 year olds oradolescents, but preferably in infants in the first year of life) thepercentage of subjects with a four-fold increase in terms of SBA(antibody dilution) titre (compared with pre-vaccination titre) againstthree heterologous strains of meningococcus is greater than 20%,preferably greater than 30%, more preferably greater than 35%, and mostpreferably greater than 40% of the subjects. Such a test is a goodindication of whether the antigen or bleb preparation with heterologousbactericidal activity can induce cross-bactericidal antibodies againstvarious meningococcal strains. The three heterologous strains shouldpreferably have different electrophoretic type (ET)-complex ormultilocus sequence typing (MLST) pattern (see Maiden et al. PNAS USA1998, 95:3140-5) to each other and preferably to the strain from whichthe antigen or bleb preparation with heterologous bactericidal activityis made or derived. A skilled person will readily be able to determinethree strains with different ET-complex which reflect the geneticdiversity observed amongst meningococci, particularly amongstmeningococcus type B strains that are recognised as being the cause ofsignificant disease burden and/or that represent recognised MenBhyper-virulent lineages (see Maiden et al. supra). For instance threestrains that could be used are the following: BZ10 (B:2b:P1.2) belongingto the A-4 cluster; B16B6 (B:2a:P1.2) belonging to the ET-37 complex;and H44/76 (B:15:P1.7, 16) belonging to the ET-5 complex, or any otherstrains belonging to the same ET/Cluster. Such strains may be used fortesting an antigen or bleb preparation with heterologous bactericidalactivity made or derived from, for instance, meningococcal strain CU385(B:4:P1.15) which belongs to the ET-5 complex. Another sample strainthat could be used is from the Lineage 3 epidemic clone (e.g. NZ124[B:4:P1.7, 4]). Another ET-37 strain is NGP165 (B:2a:P1.2).

Processes for measuring SBA activity are known in the art. For instancea method that might be used is described in WO 99/09176 in Example 10C.In general terms, a culture of the strain to be tested is grown(preferably in conditions of iron depletion—by addition of an ironchelator such as EDDA to the growth medium) in the log phase of growth.This can be suspended in a medium with BSA (such as Hanks medium with0.3% BSA) in order to obtain a working cell suspension adjusted toapproximately 20000 CFU/ml. A series of reaction mixes can be mademixing a series of two-fold dilutions of sera to be tested (preferablyheat-inactivated at 56° C. for 30 min) [for example in a 50n−1/wellvolume] and the 20000 CFU/ml meningococcal strain suspension to betested [for example in a 25 μl/well volume]. The reaction vials shouldbe incubated (e.g. 37° C. for 15 minutes) and shaken (e.g. at 210 rpm).The final reaction mixture [for example in a 100 μl volume] additionallycontains a complement source [such as 25% final volume of pretested babyrabbit serum], and is incubated as above [e.g. 37° C. for 60 min] Asterile polystyrene U-bottom 96-well microtiter plate can be used forthis assay. A aliquot [e.g. 10 μl] can be taken from each well using amultichannel pipette, and dropped onto Mueller-Hinton agar plates(preferably containing 1% Isovitalex and 1% heat-inactivated HorseSerum) and incubated (for example for 18 hours at 37° C. in 5% CO₂).Preferably, individual colonies can be counted up to 80 CFU per aliquot.The following three test samples can be used as controls:buffer+bacteria+complement; buffer+bacteria+inactivated complement;serum+bacteria+inactivated complement. SBA titers can bestraightforwardly calculated using a program which processes the data togive a measurement of the dilution which corresponds to 50% of cellkilling by a regression calculation.

Animal Protection Assays

Alternatively, the synergistic response may be characterised by theefficacy of the combination of antigens in an animal protection assay.For instance, the assays described in example 12 or 13 may be used.Preferably the number of animals protected by the combination ofantigens is significantly improved compared with using the antigens bythemselves, particularly at suboptimal doses of antigen.

A successful vaccine for the prevention of infection by N. gono mayrequire more than one of the following elements: generation of serumand/or mucosal antibodies to facilitate complement mediated killing ofthe gonococcus, and/or to enhance phagocytosis and microbial killing byleukocytes such as polymorphonuclear leukocytes, and/or to preventattachment of the gonococci to the host tissues; induction of a cellmediated immune response may also participate to protection.

The improvement of efficacy of a bleb gono vaccine preparation of theinvention can be evaluated by analyzing the induced immune response forserum and/or mucosal antibodies that have antiadherence, and/oropsonizing properties, and/or bactericidal activity, as described byothers (McChesney D et al, Infect. Immun 36: 1006, 1982; Boslego J etal: Efficacy trial of a purified gonococcl pilus vaccine, in Program andAbstracts of the 24th Interscience Conference on Antimicrobial Agentsand Chemotherapy, Washington, American Society for Microbiology, 1984;Siegel M et al, J. Infect. Dis 145: 300, 1982; de la Pas, Microbiology,141 (Pt4): 913-20, 1995).

A mouse model of genital infection by N. gono has recently beendescribed (Plante M, J. Infect. Dis., 182: 848-55, 2000). Theimprovement of efficiency of a bleb gono vaccine of the invention couldalso be evaluated by its ability to prevent or to reduce colonization byN. gono in this mouse model of infection.

Adhesion Blocking Assay

Alternatively, the synergisic response may be characterised by theefficacy of the combination of anigens in an adhesion blocking assay.For instance, the assay described in example 11 may be used. Preferablythe extent of blocking induced by antisera raised against thecombination of antigens is significantly improved compared with usingantisera raised against the antigens by themselves, particularly atsuboptimal doses of antibody.

Subunit Compositions

The immunogenic composition of the invention may be a subunitcomposition.

Subunit compositions are compositions in which the components have beenisolated and purified to at least 50%, preferably at least 60%_(,)70%_(,) 80%, 90% pure before mixing the components to form the antigeniccomposition.

The immunogenic subunit composition of the invention preferablycomprises at least 2 antigens selected from the following list: FhaB,PilC, Hsf, Hap, NadA, OMP85, IgA protease, AspA, passenger domain ofAspA, passenger domain of Hsf, passenger domain of Hap, FrpA, FrpC,TbpA, TbpB, LbpA, LbpB, HpuA, HpuB, TspA, TspB, PldA, PilQ, FhaC, NspA,and either or both of LPS immunotype L2 and LPS immunotype L3.

Subunit compositions may be aqueous solutions of water soluble proteins.They may comprise detergent, preferably non-ionic, zwitterionic or ionicdetergent in order to solubilise hydrophobic portions of the antigens.They may comprise lipids so that liposome structures could be formed,allowing presentation of antigens with a structure that spans a lipidmembrane.

Outer Membrane Vesicle Preparations

N. meningitidis serogroup B (menB) excretes outer membrane blebs insufficient quantities to allow their manufacture on an industrial scale.An outer membrane vesicles may also be prepared via the process ofdetergent extraction of the bacterial cells (see for example EP 11243).

The immunogenic composition of the invention may also comprise an outermembrane vesicle preparation having at least two antigens which havebeen upregulated, either recombinantly or by other means includinggrowth under iron-depleted conditions. Examples of antigens which wouldbe upregulated in such a outer membrane vesicle preparation include;NspA, Hsf, Hap, OMP85, TbpA (high), TbpA (low), LbpA, TbpB, LbpB, PilQ,AspA, TdfH, PorB, HpuB, P2086, NM-ADPRT, MafA, MafB and PldA. Suchpreparations would optionally also comprise either or both of LPSimmunotype L2 and LPS immunotype L3.

The manufacture of bleb preparations from Neisserial strains may beachieved by any of the methods well known to a skilled person.Preferably the methods disclosed in EP 301992, U.S. Pat. No. 5,597,572,EP 11243 or U.S. Pat. No. 4,271,147, Frederikson et al. (NIPH Annals[1991], 14:67-80), Zollinger et al. (J. Clin. Invest. [1979],63:836-848), Saunders et al. (Infect. Immun. [1999], 67:113-119),Drabick et al. (Vaccine [2000], 18:160-172) or WO 01/09350 (Example 8)are used. In general, OMVs are extracted with a detergent, preferablydeoxycholate, and nucleic acids are optionally removed enzymatically.Purification is achieved by ultracentrifugation optionally followed bysize exclusion chromatography. If 2 or more different blebs of theinvention are included, they may be combined in a single container toform a multivalent preparation of the invention (although a preparationis also considered multivalent if the different blebs of the inventionare separate compositions in separate containers which are administeredat the same time [the same visit to a practitioner] to a host). OMVpreparations are usually sterilised by filtration through a 0.2 μmfilter, and are preferably stored in a sucrose solution (e.g. 3%) whichis known to stabilise the bleb preparations.

Upregulation of proteins within outer membrane vesicle preparations maybe achieved by insertion of an extra copy of a gene into the Neisserialstrain from which the OMV preparation is derived. Alternatively, thepromoter of a gene can be exchanged for a stronger promoter in theNeisserial strain from which the OMV preparation is derived. Suchtechniques are described in WO01/09350. Upregulation of a protein willlead to a higher level of protein being present in OMV compared to thelevel of protein present in OMV derived from unmodified N. meningitidis(for instance strain H44/76). Preferably the level will be 1.5, 2, 3, 4,5, 7, 10 or 20 times higher.

Where LPS is intended to be an additional antigen in the OMV, a protocolusing a low concentration of extracting detergent (for exampledeoxycholate or DOC) may preferably be used in the OMV preparationmethod so as to preserve high levels of bound LPS whilst removingparticularly toxic, poorly bound LPS. The concentration of DOC used ispreferably 0-0.5% DOC, 0.02-0.4% DOC, 0.04-0.3% DOC more preferably0.06%-0.2% DOC or 0.08-0.15% DOC most preferably around or exactly 0.1%DOC.

“Stronger promoter sequence” refers to a regulatory control element thatincreases transcription for a gene encoding antigen of interest.

“Upregulating expression” refers to any means to enhance the expressionof an antigen of interest, relative to that of the non-modified (i.e.,naturally occurring) bleb. It is understood that the amount of‘upregulation’ will vary depending on the particular antigen of interestbut will not exceed an amount that will disrupt the membrane integrityof the bleb. Upregulation of an antigen refers to expression that is atleast 10% higher than that of the non-modified bleb. Preferably it is atleast 50% higher. More preferably it is at least 100% (2 fold) higher.Most preferably it is 3, 4, 5, 7, 10, 20 fold higher. Alternatively oradditionally, upregulating expression may refer to rendering expressionnon-conditional on metabolic or nutritional changes, particularly in thecase of TbpA, TbpB, LbpA and LbpB. Preferably the level of expression isassessed when blebs have been derived from bacteria grown in ironlimited conditions (for instance in the presence of an iron chelator).

Again for the purpose of clarity, the terms ‘engineering a bacterialstrain to produce less of said antigen’ or down regulation refers to anymeans to reduce the expression of an antigen (or the expression of afunctional gene product) of interest, relative to that of thenon-modified (i.e., naturally occurring bleb), preferably by deletion,such that expression is at least 10% lower than that of the non-modifiedbleb. Preferably it is at least 50% lower and most preferably completelyabsent. If the down regulated protein is an enzyme or a functionalprotein, the downregulation may be achieved by introducing one or moremutations resulting in a 10%, 20%, 50%, 80% or preferably a 100%reduction in enzymatic or functional activity.

The engineering steps required to modulate the expression of Neisserialproteins can be carried out in a variety of ways known to the skilledperson. For instance, sequences (e.g. promoters or open reading frames)can be inserted, and promoters/genes can be disrupted by the techniqueof transposon insertion. For instance, for upregulating a gene'sexpression, a strong promoter could be inserted via a transposon up to 2kb upstream of the gene's initiation codon (more preferably 200-600 bpupstream, most preferably approximately 400 bp upstream). Point mutationor deletion may also be used (particularly for down-regulatingexpression of a gene).

Such methods, however, may be quite unstable or uncertain, and thereforeit is preferred that the engineering step is performed via a homologousrecombination event. Preferably, the event takes place between asequence (a recombinogenic region) of at least 30 nucleotides on thebacterial chromosome, and a sequence (a second recombinogenic region) ofat least 30 nucleotides on a vector transformed within the strain.Preferably the regions are 40-1000 nucleotides, more preferably 100-800nucleotides, most preferably 500 nucleotides). These recombinogenicregions should be sufficiently similar that they are capable ofhybridising to one another under highly stringent conditions.

Methods used to carry out the genetic modification events hereindescribed (such as the upregulation or downregulation of genes byrecombination events and the introduction of further gene sequences intoa Neisserial genome) are described in WO01/09350. Typical strongpromoters that may be integrated in Neisseria are porA, porB, lgtF, Opa,p110, lst, and hpuAB. PorA and PorB are preferred as constitutive,strong promoters. It has been established that the PorB promoteractivity is contained in a fragment corresponding to nucleotides −1 to−250 upstream of the initation codon of porB.

Upregulation of Expression of Antigens by Growth in Iron LimitationMedia

The upregulation of some antigens in an outer membrane vesiclepreparation of the invention is preferably achieved by isolating outermembrane vesicles from a parental strain of Neisseria grown under ironlimitation conditions. A low concentration of iron in the medium willresult in increased expression of proteins involved in iron acquisitionincluding TbpA, TbpB, LbpA, LbpB, HpuA, HpuB and P2086. The expressionof these proteins is thereby upregulated without the need forrecombinantly modifying the gene involved, for instance by inserting astronger promoter or inserting an additional copy of the gene. Theinvention would also encompass upregulation of iron acquisition proteinsby growth in iron limitation medium where the gene has also beenrecombinantly modified.

Iron limitation is achieved by the addition of an iron chelator to theculture medium. Suitable iron chelators include 2,2-Dipyridil, EDDHA(ethylenediamine-di(o-hydroxyphenylacetic acid) and Desferal(deferoxamine mesylate, Sigma). Desferal is the preferred iron chelatorand is added to the culture medium at a concentration of between 10 and100 μM, preferably 25-75 μM, more preferably 50-70 μM, most preferablyat 60 μM. The iron content of medium comes primarily from the yeastextract and soy peptone constituents and the amount present may varybetween batches. Therefore different concentrations of Desferal may beoptimal to achieve upregulation of iron acquisition proteins indifferent batches of medium. The skilled artisan should easily be ableto determine the optimal concentration. In basic terms, enough ironchelator should be added to the medium to upregulate the expression ofthe desired iron-regulated protein, but not so much so as to adverselyaffect the growth of the bacteria.

Preferably, upregulation of iron acquisition proteins by growth underiron limited conditions is combined with recombinant upregulation ofother antigens so that the outer membrane vesicle of the invention isachieved.

Down Regulation/Removal of Variable and Non-Protective ImmunodominantAntigens

Many surface antigens are variable among bacterial strains and as aconsequence are protective only against a limited set of closely relatedstrains. An aspect of this invention covers outer membrane vesicles ofthe invention in which the expression of other proteins is reduced, or,preferably, gene(s) encoding variable surface protein(s) are deleted.Such deletion results in a bacterial strain producing blebs which, whenadministered in a vaccine, have a stronger potential forcross-reactivity against various strains due to a higher influenceexerted by conserved proteins (retained on the outer membranes) on thevaccinee's immune system. Examples of such variable antigens inNeisseria that may be downregulated in the bleb immunogenic compositionsof the invention include PorA, PorB, Opa.

Other types of gene that could be down-regulated or switched off aregenes which, in vivo, can easily be switched on (expressed) or off bythe bacterium. As outer membrane proteins encoded by such genes are notalways present on the bacteria, the presence of such proteins in thebleb preparations can also be detrimental to the effectiveness of thevaccine for the reasons stated above. A preferred example to todown-regulate or delete is Neisseria Opc protein. Anti-Opc immunityinduced by an Opc containing bleb vaccine would only have limitedprotective capacity as the infecting organism could easily become Opc⁻.

For example, these variable or non-protective genes may bedown-regulated in expression, or terminally switched off. This has theadvantage of concentrating the immune system on better antigens that arepresent in low amounts on the outer surface of blebs. By down-regulationit is also meant that surface exposed, variable immunodominant loops ofthe above outer membrane proteins may be altered or deleted in order tomake the resulting outer membrane protein less immunodominant.

Methods for downregulation of expression are disclosed in WO01/09350.Preferred combinations of proteins to be downregulated in the blebimmunogenic compositions of the invention include PorA and OpA; PorA andOpC; OpA and OpC; PorA and OpA and OpC.

Four different Opa genes are known to exist in the meningococcal genome(Aho et al. 1991 Mol. Microbiol. 5:1429-37), therefore where Opa is saidto be downregulated in expression it is meant that preferably 1, 2, 3 or(preferably) all 4 genes present in meningococcus are so downregulated.Such downregulation may be performed genetically as described in WO01/09350 or by seeking readily-found, natural, stable meningococcalstrains that have no or low expression from the Opa loci. Such strainscan be found using the technique described in Poolman et al (1985 J.Med. Micro. 19:203-209) where cells that are Opa⁻ have a differentphenotype to cells expressing Opa which can be seen looking at theappearance of the cells on plates or under a microscope. Once found, thestrain can be shown to be stably Opa⁻ by performing a Western blot oncell contents after a fermentation run to establish the lack of Opa.

Where upregulation of some antigens in the outer membrane vesicle isachieved by growth under iron limitation conditions, the variableprotein FrpB (Microbiology 142; 3269-3274, (1996); J. Bacteriol. 181;2895-2901 (1999)) will also be upregulated. The inventors have foundthat it is advantageous to down-regulate expression of FrpB under thesecircumstances by downregulating expression of the entire protein asdescribed in WO01/09350 or by deleting variable region(s) of FrpB. Thiswill ensure that the immune response elicited by the immunogeniccomposition is directed towards antigens that are present in a widerange of strains. Down regulation of FrpB is preferably combined withdown regulation of PorA and OpA; PorA and OpC; OpA and OpC; PorA and OpAand OpC in the bleb immunogenic compositions of the invention.

In an alternative embodiment of the invention, FrpB is downregulated inouter membrane vesicles which have been prepared from Neisseria strainsnot grown under iron limitation conditions.

Detoxification of LPS

The blebs in the immunogenic compositions of the invention may bedetoxified via methods for detoxification of LPS which are disclosed inWO01/09350. In particular methods for detoxification of LPS of theinvention involve the downregulation/deletion of htrB and/or msbBenzymes which are disclosed in WO01/09350. The msbB and htrB genes ofNeisseria are also called 1pxL1 and 1pxL2, respectively (WO 00/26384)and deletion mutationsof these genes are characterised pnenoltypicallyby the msbB-mutant LOS losing one secondary acyl chain), and thehtrB-mutatn LOS losing both secondary acyl chains. WO93/14155 and WO95/03327 describe nontoxix peptide functional equivalents of polymycin Bthat may be used in compositions of the invention.

Such methods are preferably combined with methods of bleb extractioninvolving low levels of DOC, preferably 0-0.3% DOC, more preferably0.05%-0.2% DOC, most preferably around or exactly 0.1% DOC.

Further methods of LPS detoxification include adding to the blebpreparations a non-toxic peptide functional equivalent of polymyxin B(preferably SAEP 2) as described above.

Cross-Reactive Polysaccharides

The isolation of bacterial outer-membrane blebs from encapsulatedGram-negative bacteria often results in the co-purification of capsularpolysaccharide. In some cases, this “contaminant” material may proveuseful since polysaccharide may enhance the immune response conferred byother bleb components. In other cases however, the presence ofcontaminating polysaccharide material in bacterial bleb preparations mayprove detrimental to the use of the blebs in a vaccine. For instance, ithas been shown at least in the case of N. meningitidis that theserogroup B capsular polysaccharide does not confer protective immunityand is susceptible to induce an adverse auto-immune response in humans.Consequently, outer membrane vesicles of the invention may be isolatedfrom a bacterial strain for bleb production, which has been engineeredsuch that it is free of capsular polysaccharide. The blebs will then besuitable for use in humans. A particularly preferred example of such ableb preparation is one from N. meningitidis serogroup B devoid ofcapsular polysaccharide.

This may be achieved by using modified bleb production strains in whichthe genes necessary for capsular biosynthesis and/or export have beenimpaired. Inactivation of the gene coding for capsular polysaccharidebiosynthesis or export can be achieved by mutating (point mutation,deletion or insertion) either the control region, the coding region orboth (preferably using the homologous recombination techniques describedabove), or by any other way of decreasing the enzymatic function of suchgenes. Moreover, inactivation of capsular biosynthesis genes may also beachieved by antisense over-expression or transposon mutagenesis. Apreferred method is the deletion of some or all of the Neisseriameningitidis cps genes required for polysaccharide biosynthesis andexport. For this purpose, the replacement plasmid pMF121 (described inFrosh et a1.1990, Mol. Microbiol. 4:1215-1218) can be used to deliver amutation deleting the cpsCAD (+galE) gene cluster.

The safety of antibodies raised to L3 or L2 LPS has been questioned, dueto the presence of a structure similar to the lacto-N-neotetraoseoligosaccharide group (Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-) present in humanglycosphingolipids. Even if a large number of people has been safelyvaccinated with deoxycholate extracted vesicle vaccines containingresidual amount of L3 LPS (G. Bjune et al, Lancet (1991), 338,1093-1096; GVG. Sierra et al, NIPH ann (1991), 14, 195-210), thedeletion of the terminal part of the LOS saccharidic is advantageous inpreventing any cross-reaction with structures present at the surface ofhuman tissues. In a preferred embodiment, inactivation of the lgtB generesults in an intermediate LPS structure in which the terminal galactoseresidue and the sialic acid are absent (the mutation leaves a4GlcNAcβ1-3Galβ1-4Glcβ1- structure in L2 and L3 LOS). Such intermediatescould be obtained in an L3 and an L2 LPS strain. An alternative and lesspreferred (short) version of the LPS can be obtained by turning off thelgtE gene. A further alternative and less preferred version of the LPScan be obtained by turning off the lgtA gene. If such an lgtA⁻ mutationis selected it is preferred to also turn off lgtC expression to preventthe non-immunogenic L1 immunotype being formed.

LgtB⁻ mutants are most preferred as the inventors have found that thisis the optimal truncation for resolving the safety issue whilst stillretaining an LPS protective oligosaccharide epitope that can stillinduce a bactericidal antibody response.

Therefore, immunogenic compositions of the invention further comprisingL2 or L3 preparations (whether purified or in an isolated bleb) ormeningococcal bleb preparations in general are advantageously derivedfrom a Neisserial strain (preferably meningococcal) that has beengenetic engineered to permanently downregulate the expression offunctional gene product from the lgtB, lgtA or lgtE gene, preferably byswitching the gene off, most preferably by deleting all or part of thepromoter and/or open-reading frame of the gene.

Where the above immunogenic compositions of the invention are derivedfrom a meningococcus B strain, it is further preferred that the capsularpolysaccharide (which also contains human-like saccharide structures) isalso removed. Although many genes could be switched off to achieve this,the inventors have advantageously shown that it is preferred that thebleb production strain has been genetically engineered to permanentlydownregulate the expression of functional gene product from the siaDgene (i.e. downregulating α-2-8 polysialyltransferase activity),preferably by switching the gene off, most preferably by deleting all orpart of the promoter and/or open-reading frame of the gene. Such aninactivation is described in WO 01/09350. The siaD (also known as synD)mutation is the most advantageous of many mutations that can result inremoving the human-similar epitope from the capsular polysaccharide,because it one of the only mutations that has no effect on thebiosynthesis of the protective epitopes of LOS, thus being advantageousin a process which aims at ultimately using LOS as a protective antigen,and has a minimal effect on the growth of the bacterium. A preferredaspect of the invention is therefore a bleb immunogenic preparation asdescribed above which is derived from an lgtE⁻ siaD⁻, an lgtA⁻ siaD⁻ or,preferably, an lgtB⁻ siaD⁻ meningococcus B mutant strain. The strainitself is a further aspect of the invention.

Although siaD⁻ mutation is preferable for the above reasons, othermutations which switch off meningococcus B capsular polysaccharidesynthesis may be used. Thus bleb production strain can be geneticallyengineered to permanently downregulate the expression of functional geneproduct from one or more of the following genes: ctrA, ctrB, ctrC, ctrD,synA (equivalent to synX and siaA), synB (equivalent to siaB) or synC(equivalent to siaC) genes, preferably by switching the gene off, mostpreferably by deleting all or part of the promoter and/or open-readingframe of the gene. The lgtE⁻ mutation may be combined with one or moreof these mutations. Preferably the lgtB⁻ mutation is combined with oneor more of these mutations. A further aspect of the invention istherefore a bleb immunogenic preparation as described above which isderived from such a combined mutant strain of meningococcus B. Thestrain itself is a further aspect of the invention.

A Neisserial locus containing various lgt genes, including lgtB andlgtE, and its sequence is known in the art (see M. P. Jennings et al,Microbiology 1999, 145, 3013-3021 and references cited therein, and J.Exp. Med. 180:2181-2190 [1994]).

Where full-length (non-truncated) LOS is to be used in the finalproduct, it is desirable for LOS not to be sialyated (as such LOSgenerates an immune response against the most dangerous, invasivemeningococcal B strains which are also unsialylated). In such case usinga capsule negative strain which has a deleted synA (equivalent to synXand siaA), synB (equivalent to siaB) or synC (equivalent to siaC) geneis advantageous, as such a mutation also renders menB LOS incapable ofbeing sialylated.

In bleb preparations, particularly in preparations extracted with lowDOC concentrations LPS may be used as an antigen in the immunogeniccomposition of the invention. It is however advantageous todownregulate/delete/inactivate enzymatic function of either the lgtE,lgtA (particularly in combination with lgtC), or, preferably, lgtBgenes/gene products in order to remove human like lacto-N-neotetraosestructures. The Neisserial locus (and sequence thereof) comprising thelgt genes for the biosynthesis of LPS oligosaccharide structure is knownin the art (Jennings et al Microbiology 1999 145; 3013-3021 andreferences cited therein, and J. Exp. Med. 180:2181-2190 [1994]).Downregulation/deletion of lgtB (or functional gene product) ispreferred since it leaves the LPS protective epitope intact.

In N. meningitidis serogroup B bleb preparations of the invention, thedownregulation/deletion of both siaD and lgtB is preferred, (although acombination of lgtB⁻ with any of ctrA⁻, ctrB⁻, ctrC⁻, ctra, synA⁻(equivalent to synX⁻ and siaK), synB⁻ (equivalent to siaB⁻) or synC⁻(equivalent to siaC⁻) in a meningococcus B bleb production strain mayalso be used) leading to a bleb preparation with optimal safety and LPSprotective epitope retention.

A further aspect of the invention is therefore a bleb immunogenicpreparation as described above which is derived from such a combinedmutant strain of meningococcus B. The strain itself is a further aspectof the invention.

Immunogenic composition of the invention may comprise at least, one,two, three, four or five different outer membrane vesicle preparations.Where two or more OMV preparations are included, at least one antigen ofthe invention is upregulated in each OMV. Such OMV preparations may bederived from Neisserial strains of the same species and serogroup orpreferably from Neisserial strains of different class, serogroup,serotype, subserotype or immunotype. For example, an immunogeniccomposition may comprise one or more outer membrane vesiclepreparation(s) which contains LPS of immunotype L2 and one or more outermembrane vesicle preparation which contains LPS of immunotype L3. L2 orL3 OMV preparations are preferably derived from a stable strain whichhas minimal phase variability in the LPS oligosaccharide synthesis genelocus.

Outer Membrane Vesicles Combined with Subunit Compositions

The immunogenic compositions of the invention may also comprise both asubunit composition and an outer membrane vesicle. There are severalantigens that are particularly suitable for inclusion in a subunitcomposition due to their solubility. Examples of such proteins include;FhaB, NspA, passenger domain of Hsf, passenger domain of Hap, passengerdomain of AspA, AspA, OMP85, FrpA, FrpC, TbpB, LbpB, PilQ. The outermembrane vesicle preparation would have at least one different antigenselected from the following list which has been recombinantlyupregulated in the outer membrane vesicle: NspA, Hsf, Hap, OMP85, TbpA(high), TbpA (low), LbpA, TbpB, LbpB, NadA, TspA, TspB, PilC, PilQ,TdfH, PorB, HpuB, P2086, NM-ADPRT, MafA, MafB and PldA; and optionallycomprise either or both of LPS immunotype L2 and LPS immunotype L3.

Specific Immunogenic Compositions of the Invention

In the specific combinations listed below, where combinations ofantigens are present in a bleb, such combinations of antigens should beupregulated as descibed above.

A particularly preferred embodiment of the invention comprises anautotransporter protein and an iron acquisition protein, more preferablyHsf and TbpA (high) and/or TbpA (low). Such immunogenic compositions maymore preferably further comprise at least one of OMP 85, FrpA, FrpC,LbpA, LbpB, Lipo28, Sibp, NMB0964, NMB0293, TspA, NadA, TspB, PilQ,FhaC, NspA, PldA, HimD, HisD, GNA1870, OspA, HlpA, FhaB, PilC, Omp26,NMB0315, NMB0995, NMB1119, TdfH, PorB, HpuB, P2086, NM-ADPRT, VapD andHap. All the above immunogenic compositions may further comprise eitheror both of LPS immunotype L2 and LPS immunotype L3.

A further preferred embodiment of the invention comprises Hsf and atleast one further antigen selected form the group consisting of FrpA,FrpC,NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, P2086, HpuA, HpuB, Lipo28,Sibp, Hap, AspA, IgA protease, OMP85, NspA, PilQ, HimD, HisD, GNA1870,OspA, HlpA, FhaC, NadA, PldA, TspA, TspB, TdfH, PorB and FhaB. All theabove immunogenic compositions may further comprise either or both ofLPS immunotype L2 and LPS immunotype L3. Preferred combinations compriseHsf and OMP85 (optionally with one or more of Hap, FrpA or LbpB); Hsfand Hap(optionally with one or more of FrpA, LbpB or OMP85); Hsf andFrpA (optionally with one or more of Hap, LbpB or OMP85); Hsf and LbpB(optionally with one or more of Hap, OMP85 or FrpA). In as much as Hsfis an adhesin and an autotransporter protein, a particularly preferredcombination comprises Hsf, OMP85, TbpA, LPS immunotype L2 and/or L3,preferably in a multivalent bleb preparation, which has members of allfive groups of antigens represented. Preferably both TbpA(low) andTbpA(high) are present.

A further immunogenic composition of the invention comprises FhaB and atleast one further antigen selected from the group consisting of FrpA,FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, HpuA, HpuB, P2086, Lipo28, Sibp,NMB0964, NMB0293, TdfH, PorB, PldA, Hap, IgA protease, AspA, PilQ, HimD,HisD, GNA1870, OspA, HlpA, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995,NMB 1119, NadA, PldA, TbpA, Hsf, TspA and TspB, and either or both ofLPS immunotype L2 and LPS immunotype L3. Preferred combinations compriseFhaB and Hsf (optionally with one or more of OMP85, LbpB, Hap or FrpA);FhaB and OMP85 (optionally with one or more of LbpB, Hap or FrpA); FhaBand LbpB (optionally with one or more of Hap or FrpA); FhaB and Hap(optionally with FrpA). A preferred combination comprises FhaB, LbpB,Hsf (as an OMP) and FrpA which has members of all five groups of antigenrepresented.

A further immunogenic composition of the invention comprises NspA and atleast one further antigen selected from the group consisting of FrpA,FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, HpuA, HpuB, P2086, Lipo28,Sibp, NMB0964, NMB0293, Hap, OMP85, PilQ, AspA, IgA protease, NadA,PldA, Hsf, Hap, TspA, TspB, TdfH, PorB, and either or both of LPSimmunotype L2 and LPS immunotype L3. Preferred combinations compriseNspA and Hsf (optionally with one or more of OMP85, Hap, LbpA or TbpA);NspA and OMP85 (optionally with one or more of Hap, LbpA or TbpA); NspAand Hap (optionally with one or more of LbpA or TbpA); NspA and LbpA(optionally with TbpA). A particularly preferred combination comprisesNspA, Hsf, TbpA, LPS immunotype L2 and/or L3, preferably in amultivalent bleb preparation, which has members of all five groups ofantigens represented. Preferably both TbpA(low) and TbpA(high) arepresent.

Immunogenic compositins with individualised combinatins of antigensdisclosed in WO 00/25811 are not claimed in this invention. Preferably,immunogenic compositions or vaccines are not covered by the presentinvention if they have an antigen content consisting solely oftransferrin binding protein and NspA (or in the case of a bleb vaccine,have an upregulated or enriched antigen content consisting solely oftransferrin binding protein and NspA), however specific combinations ofantigens (or upregulated antigens) consisting of or including NspA aswell as both TbpA(high) and TbpA (low) may be included. Optionally,compositions or vaccines comprising a combination (subunit) orupregulation (bleb) of transferrin binding protein and NspA are notclaimed.

A further immunogenic composition of the invention comprises NadA and atleast one further antigen selected from the group consisting of FrpA,FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, P2086, Lipo28, Sibp,NMB0964, NMB0293, Hap, OMP85, NspA, PilQ, HimD, HisD, GNA1870, OspA,HlpA, HpuA, HpuB, AspA, IgA protease, PldA, Hsf, TspA, TspB, TdfH, PorB,and either or both of LPS immunotype L2 and LPS immunotype L3.

A further immunogenic composition of the invention comprises TbpA (low)and at least one further antigen selected from the group consisting ofFrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, IgA protease, NspA, HpuA,HpuB, Hap, OMP85, NspA (when further combined with TbpA(high)), PilQ,HimD, HisD, GNA1870, OspA, HlpA, PilC, Omp26, NMB0315, NMB0995, NMB1119,MafA, MafB, AspA, NadA, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB, andeither or both of LPS immunotype L2 and LPS immunotype L3. Preferredcombinations comprise TbpA(low) and Hsf and LbpA; TbpA(low) and OMP85(optionally with either or both of LbpA and Hap); TbpA(low) and LbpA andHap.

A further immunogenic composition of the invention comprises TbpA (high)and at least one further antigen selected from the group consisting ofFrpA, FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, Hap, OMP85, NspA (whenfurther combined with TbpA(low)), PilC, Omp26, NMB0315, NMB0995,NMB1119, PilQ, HimD, HisD, GNA1870, OspA, HlpA, MafA, MafB, AspA, IgAprotease, PldA, FhaB, NadA, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB,and either or both of LPS immunotype L2 and LPS immunotype L3. Preferredcombinations comprise TbpA(high) and Hsf and LbpA; TbpA(high) and OMP85(optionally with either or both of LbpA and Hap); TbpA(high) and LbpAand Hap.

A further immunogenic composition of the invention comprises LbpA and atleast one further antigen selected from the group consisting of FrpA,FrpC, NM-ADPRT, VapD, LbpB, TbpB, Hap, OMP85, NspA, PilC, Omp26,NMB0315, NMB0995, NMB 1119, NadA, PldA, TbpA, Hsf, TspA, TspB, MafA,MafB, IgA protease, AspA, FhaB, PilQ, HimD, HisD, GNA1870, OspA, HlpA,TdfH, PorB and FhaB and either or both of LPS immunotype L2 and LPSimmunotype L3. Preferred combinations comprise LbpA and Hsf (optionallywith Hap).

A further immunogenic composition of the invention comprises LbpB and atleast one further antigen selected from the group consisting of FrpA,FrpC, NM-ADPRT, VapD, LbpA, TbpB, Hap, OMP85, NspA, PilC, Omp26,NMB0315, NMB0995, NMB 1119, NadA, PldA, TbpA, Hsf, TspA, TspB, MafA,MafB, IgA protease, AspA, FhaB, PilQ, HimD, HisD, GNA1870, OspA, HlpA,TdfH, PorB and FhaB, and either or both of LPS immunotype L2 and LPSimmunotype L3. Preferred combinations comprise LbpB and Hsf (optionallywith one or more of OMP85, Hap or FrpA); LbpB and OMP85 (optionally withone or more of Hap or FrpA); LbpB and Hap (optionally with FrpA).

A further immunogenic composition of the invention comprises OMP85 andat least one further antigen selected from the group consisting of FrpA,FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, HpuA, HpuB, P2086, Lipo28,Sibp, NMB0964, NMB0293, Hap, IgA protease, AspA, Hsf, NspA, PilC, Omp26,NMB0315, NMB0995, NMB1119, MafA, MafB, NadA, PldA, Hsf, TspA, TspB,PilQ, TdfH, PorB and FhaB, and either or both of LPS immunotype L2 andLPS immunotype L3. Preferred combinations comprise OMP85 and Hsf(optionally with either or both of LbpA or NspA); OMP85 and LbpA(optionally with either or both of Hap and NspA); OMP85 and Hap(optionally with NspA).

A further immunogenic composition of the invention comprises Hap and atleast one further antigen selected from the group consisting of FrpA,FrpC, NM-ADPRT, VapD, LbpB, LbpA, TbpB, TbpA, HpuA, HpuB, P2086, Lipo28,Sibp, NMB0964, NMB0293, PilQ, HimD, HisD, GNA1870, OspA, HlpA, NspA, IgAprotease, AspA, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119,MafA, MafB, NadA, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB, and eitheror both of LPS immunotype L2 and LPS immunotype L3.

A further immunogenic composition of the invention comprises FrpA and atleast one further antigen selected from the group consisting of LbpB,LbpA, TbpA, TbpB, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293,PilQ, HimD, HisD, GNA1870, OspA, HlpA, TspA, TspB, Hap, IgA protease,AspA, NadA, FhaB, PilQ, HimD, HisD, GNA1870, OspA, HlpA, OMP85, NspA,PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, PldA, Hsf, TspA,TspB, TdfH, PorB and FhaB, and either or both of LPS immunotype L2 andLPS immunotype L3.

A further immunogenic composition of the invention comprises FrpC and atleast one further antigen selected from the group consisting of LbpB,LbpA, TbpA, TbpB, HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293,PilQ, HimD, HisD, GNA1870, OspA, HlpA, TspA, TspB, Hap, IgA protease,AspA, NadA, FhaB, OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119,MafA, MafB, PldA, Hsf, TspA, TspB, TdfH, PorB and FhaB, and either orboth of LPS immunotype L2 and LPS immunotype L3.

A further immunogenic composition of the invention comprises either orboth of LPS immunotype L2 and LPS immunotype L3 and at least one furtherantigen selected from the group consisting of LbpB, LbpA, TbpA, TbpB,HpuA, HpuB, P2086, Lipo28, Sibp, NMB0964, NMB0293, PilQ, HimD, HisD,GNA1870, OspA, HlpA, TspA, TspB, Hap, IgA protease, AspA, NadA, FhaB,OMP85, NspA, PilC, Omp26, NMB0315, NMB0995, NMB1119, MafA, MafB, PldA,Hsf, TspA, TspB, TdfH, PorB and FhaB.

Preferred combinations of antigens in an immunogenic composition of theinvention include combinations comprising an iron acquisition protein,an autotransporter protein and FhaB; an iron acquisition protein, anautotransporter protein and PilC; an iron acquisition protein, anautotransporter protein and NadA; an iron acquisition protein, anautotransporter protein and FrpA; an iron acquisition protein, anautotransporter protein and PilQ; an iron acquisition protein, anautotransporter protein and TspA; an iron acquisition protein, anautotransporter protein and TspB; an iron acquisition protein, anautotransporter protein and NspA; an iron acquisition protein, anautotransporter protein and FrpC; more preferably comprising an ironacquisition protein, an autotransporter protein and Hap; an ironacquisition protein, an autotransporter protein and FrpA/C; an ironacquisition protein, an autotransporter protein and LbpB; an ironacquisition protein, an autotransporter protein and OMP85 (D15). Mostpreferably, OMP85 (D15) would be incorporated as part of an outermembrane vesicle preparation.

Immunogenic compositions of the invention which contain LPS willpreferably have the LPS conjugated to a source of T-helper epitopes,preferably proteins, and in the case of LPS in OMVs, preferably outermembrane proteins. A particularly preferred embodiment contains LPSwhich have been (preferably intra-bleb) conjugated to OMP in situ in theouter membrane vesicle preparation (for instance as described above).

The immunogenic compositions of the invention may comprise antigens(proteins, LPS and polysaccharides) derived from Neisseria meningitidisserogroups A, B, C, Y, W-135 or Neisseria gonorrhoeae.

Preferably the immunogenic compositions or vaccines of the invention donot consist of and/or comprise the particular combinations of SEQ IDslisted in the table spanning from page 3, line 18 to page 52, line 2 ofWO 00/71725 and/or any individual combination described in the examples1-11 of WO 00/71725.

Preferably, any individualised combinations disclosed in WO 01/52885 arenot claimed in this invention.

Further Combinations

The immunogenic composition of the invention may further comprisebacterial capsular polysaccharides or oligosaccharides. The capsularpolysaccharides or oligosaccharides may be derived from one or more of:Neisseria meningitidis serogroup A, C, Y, and/or W-135, Haemophilusinfluenzae b, Streptococcus pneumoniae, Group A Streptococci, Group BStreptococci, Staphylococcus aureus and Staphylococcus epidermidis.

A further aspect of the invention are vaccine combinations comprisingthe antigenic composition of the invention with other antigens which areadvantageously used against certain disease states including thoseassociated with viral or Gram positive bacteria.

In one preferred combination, the antigenic compositions of theinvention are formulated with 1, 2, 3 or preferably all 4 of thefollowing meningococcal capsular polysaccharides or oligosaccharideswhich may be plain or conjugated to a protein carrier: A, C, Y or W-135.Preferably the immunogenic compositions of the invention are formulatedwith A and C; or C; or C and Y. Such a vaccine containing proteins fromN. meningitidis, preferably serogroup B may be advantageously used as aglobal meningococcus vaccine.

In a further preferred embodiment, the antigenic compositions of theinvention, preferably formulated with 1, 2, 3 or all 4 of the plain orconjugated meningococcal capsular polysaccharides or oligosaccharides A,C, Y or W-135 (as described above), are formulated with a conjugated H.influenzae b capsular polysaccharide or oligosaccharides, and/or one ormore plain or conjugated pneumococcal capsular polysaccharides oroligosaccharides. Optionally, the vaccine may also comprise one or moreprotein antigens that can protect a host against Streptococcuspneumoniae infection. Such a vaccine may be advantageously used as aglobal meningitis vaccine.

In a still further preferred embodiment, the immunogenic composition ofthe invention is formulated with capsular polysaccharides oroligosaccharides derived from one or more of Neisseria meningitidis,Haemophilus influenzae b, Streptococcus pneumoniae, Group AStreptococci, Group B Streptococci, Staphylococcus aureus orStaphylococcus epidermidis. The pneumococcal capsular polysaccharideantigens are preferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F,8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19Fand 23F). A further preferred embodiment would contain the PRP capsularpolysaccharides of Haemophilus influenzae. A further preferredembodiment would contain the Type 5, Type 8 or 336 capsularpolysaccharides of Staphylococcus aureus. A further preferred embodimentwould contain the Type I, Type II or Type III capsular polysaccharidesof Staphylococcus epidermidis. A further preferred embodiment wouldcontain the Type Ia, Type Ic, Type II or Type III capsularpolysaccharides of Group B streptocoocus. A further preferred embodimentwould contain the capsular polysaccharides of Group A streptococcus,preferably further comprising at least one M protein and more preferablymultiple types of M protein.

Such capsular polysaccharides of the invention may be unconjugated orconjugated to a carrier protein such as tetatus toxoid, tetanus toxoidfragment C, diphtheria toxoid, CRM197, pneumolysin, Protein D (U.S. Pat.No. 6,342,224). The polysaccharide conjugate may be prepared by anyknown coupling technique. For example the polysaccharide can be coupledvia a thioether linkage. This conjugation method relies on activation ofthe polysaccharide with 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) to form a cyanate ester. The activatedpolysaccharide may thus be coupled directly or via a spacer group to anamino group on the carrier protein. Preferably, the cyanate ester iscoupled with hexane diamine and the amino-derivatised polysaccharide isconjugated to the carrier protein using heteroligation chemistryinvolving the formation of the thioether linkage. Such conjugates aredescribed in PCT published application WO93/15760 Uniformed ServicesUniversity.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508. A further method involves the coupling of acyanogen bromide activated polysaccharide derivatised with adipic acidhydrazide (ADH) to the protein carrier by Carbodiimide condensation (ChuC. et al Infect. Immunity, 1983 245 256). Where oligosaccharides areincluded, it is preferred that they be conjugated.

Preferred pneumococcal proteins antigens are those pneumococcal proteinswhich are exposed on the outer surface of the pneumococcus (capable ofbeing recognised by a host's immune system during at least part of thelife cycle of the pneumococcus), or are proteins which are secreted orreleased by the pneumococcus. Most preferably, the protein is a toxin,adhesin, 2-component signal tranducer, or lipoprotein of Streptococcuspneumoniae, or fragments thereof. Particularly preferred proteinsinclude, but are not limited to: pneumolysin (preferably detoxified bychemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990Jul. 11; 18(13): 4010 “Comparison of pneumolysin genes and proteins fromStreptococcus pneumoniae types 1 and 2.”, Mitchell et al. BiochimBiophys Acta 1989 Jan. 23; 1007(1): 67-72 “Expression of the pneumolysingene in Escherichia coli: rapid purification and biologicalproperties.”, WO 96/05859 (A. Cyanamid), WO 90/06951 (Paton et al), WO99/03884 (NAVA)]; PspA and transmembrane deletion variants thereof (U.S.Pat. No. 5,804,193—Briles et al.); PspC and transmembrane deletionvariants thereof (WO 97/09994—Briles et al); PsaA and transmembranedeletion variants thereof (Berry & Paton, Infect Immun 1996 December;64(12):5255-62 “Sequence heterogeneity of PsaA, a 37-kilodalton putativeadhesin essential for virulence of Streptococcus pneumoniae”);pneumococcal choline binding proteins and transmembrane deletionvariants thereof; CbpA and transmembrane deletion variants thereof (WO97/41151; WO 99/51266); Glyceraldehyde-3-phosphate—dehydrogenase(Infect. Immun. 1996 64:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beatoet al. FEMS Microbiol Lett 1998, 164:207-14); M like protein, (EP0837130) and adhesin 18627, (EP 0834568). Further preferred pneumococcalprotein antigens are those disclosed in WO 98/18931, particularly thoseselected in WO 98/18930 and PCT/U599/30390.

The immunogenic composition/vaccine of the invention may also optionallycomprise outer membrane vesicle preparations made from other Gramnegative bacteria, for example Moraxella catarrhalis or Haemophilusinfluenzae.

Moraxella catarrhalis bleb preparations

Immunogenic compositions of the invention may further comprise OMVpreparations derived from Moraxella catarrhalis. Engineered OMVpreparations can be derived from Moraxella catarrhalis as described inWO01/09350. One or more of the following genes (encoding protectiveantigens) are preferred for upregulation: OMP106 (WO 97/41731 & WO96/34960), HasR(PCT/EP99/03824), PilQ (PCT/EP99/03823), OMP85(PCT/EP00/01468), lipo06 (GB 9917977.2), lipo 10 (GB 9918208.1), lipo 11(GB 9918302.2), lipol8 (GB 9918038.2), P6 (PCT/EP99/03038), ompCD, CopB(Helminen M E, et al (1993) Infect. Immun 61:2003-2010), D15(PCT/EP99/03822), Omp1A1 (PCT/EP99/06781), H1y3 (PCT/EP99/03257), LbpAand LbpB (WO 98/55606), TbpA and TbpB (WO 97/13785 & WO 97/32980), OmpE,UspA 1 and UspA2 (WO 93/03761), and Omp21. They are also preferred asgenes which may be heterologously introduced into other Gram-negativebacteria.

One or more of the following genes are preferred for downregulation:CopB, OMP 106, OmpB1, TbpA, TbpB, LbpA, and LbpB.

One or more of the following genes are preferred for downregulation:htrB, msbB and 1pxK.

One or more of the following genes are preferred for upregulation: pmrA,pmrB, pmrE, and pmrF.

Haemophilus influenzae bleb Preparations

Immunogenic compositions of the invention may further comprise OMVpreparations derived from Haemophilus influenzae. Engineered OMVpreparations can be derived from Haemophilus influenzae as described inWO01/09350. One or more of the following genes (encoding protectiveantigens) are preferred for upregulation: D15 (WO 94/12641), P6 (EP281673), TbpA (WO96/40929; WO95/13370), TbpB (WO96/40929; WO95/13370),P2, P5 (WO 94/26304), OMP26 (WO 97/01638), HMW1, HMW2, HMW3, HMW4, Hia,Hsf, Hap, Hin47, and Hif (all genes in this operon should be upregulatedin order to upregulate pilin). They are also preferred as genes whichmay be heterologously introduced into other Gram-negative bacteria.

One or more of the following genes are preferred for downregulation: P2,P5, Hif, IgA1-protease, HgpA, HgpB, HMW1, HMW2, Hxu, htrB, msbB and1pxK.

One or more of the following genes are preferred for upregulation: pmrA,pmrB, pmrE, and pmrF.

The immunogenic composition/vaccine of the invention may also optionallycomprise antigens providing protection against one or more ofDiphtheria, tetanus and Bordetella pertussis infections. The pertussiscomponent may be killed whole cell B. pertussis (Pw) or acellularpertussis (Pa) which contains at least one antigen (preferably 2 or all3) from PT, FHA and 69 kDa pertactin. Typically, the antigens providingprotection against Diphtheria and tetanus would be Diphtheria toxoid andtetanus toxoid. The toxoids may chemically inactivated toxins or toxinsinactivated by the introduction of point mutations.

The immunogenic composition/vaccine may also optionally comprise one ormore antigens that can protect a host against non-typeable Haemophillusinfluenzae, RSV and/or one or more antigens that can protect a hostagainst influenza virus. Such a vaccine may be advantageously used as aglobal otitis media vaccine.

Preferred non-typeable H. influenzae protein antigens include Fimbrinprotein (U.S. Pat. No. 5,766,608) and fusions comprising peptidestherefrom (eg LB1 Fusion) (U.S. Pat. No. 5,843,464—Ohio State ResearchFoundation), OMP26, P6, protein D, TbpA, TbpB, Hia, Hmw1, Hmw2, Hap, andD15.

Preferred influenza virus antigens include whole, live or inactivatedvirus, split influenza virus, grown in eggs or MDCK cells, or Vero cellsor whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10,915-920) or purified or recombinant proteins thereof, such as HA, NP,NA, or M proteins, or combinations thereof.

Preferred RSV (Respiratory Syncytial Virus) antigens include the Fglycoprotein, the G glycoprotein, the HN protein, the M protein orderivatives thereof.

Immunogenic compositions of the invention may include proteins ofMoraxella catarrhalis include TbpA (WO97/13785; WO99/52947), TbpB(WO97/13785; WO99/52947; Mathers et al FEMS Immunol Med Microbiol 199719; 231-236; Myers et al Infect Immun 1998 66; 4183-4192), LbpA, LbpB(Du et al Infect Immun 1998 66; 3656-3665), UspA1, UspA2 (Aebi et alInfect Immun. 1997 65; 4367-4377), OMP106 (U56214981), Ton-B dependentreceptor (WO00/78968), CopB (Sethi et al Infect. Immun. 1997 65;3666-3671), and HasR receptor (WO00/78968); proteins of Haemophilusinfluenzae include HMW (St Geme et al Infect Immun 1998 66; 364-368),Hia (St Geme et al J. Bacteriol. 2000 182; 6005-6013), Tbp1 (WO96/40929;WO95/13370), Tbp2 (WO96/40929; WO95/13370; Gray-Owen et al Infect Immun1995 63; 1201-1210), LbpA, LbpB (Schryvers et al 1989, 29:121-130),HasR, TonB-dependent receptor (Fleishmann et al Science 1995 269;496-512), hemoglobin-binding protein, HhuA (Cope et al Infect Immun 200068; 4092-4101), HgpA (Maciver et al Infect Immun 1996 64; 3703-3712),HgbA, HgbB and HgbC (Jin et al Infect Immun 1996 64; 3134-3141), HxuA(Cope et al Mol Microbiol 1994 13; 863-873), HxuC (Cope et al InfectImmun 2001 69; 2353-2363); proteins from Neisseria meningitidis includeTbp1, Tbp2, FbpA, FbpB, BfrA, BfrB (Tettelin et al Science 2000 287;1809-1815), LbpA, LbpB and HmbR.

Vaccine Formulations

A preferred embodiment of the invention is the formulation of theimmunogenic composition of the invention in a vaccine which may alsocomprise a pharmaceutically acceptable excipient or carrier.

The manufacture of outer membrane vesicle preparations from any of theaforementioned modified strains may be achieved by any of the methodswell known to a skilled person. Preferably the methods disclosed in EP301992, U.S. Pat. No. 5,597,572, EP 11243 or U.S. Pat. No. 4,271,147 areused. Most preferably, the method described in WO 01/09350 is used.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M.F. & Newman M.J.) (1995)Plenum Press New York).

The antigenic compositions of the present invention may be adjuvanted inthe vaccine formulation of the invention. Suitable adjuvants include analuminium salt such as aluminum hydroxide gel (alum) or aluminiumphosphate, but may also be a salt of calcium (particularly calciumcarbonate), iron or zinc, or may be an insoluble suspension of acylatedtyrosine, or acylated sugars, cationically or anionically derivatisedpolysaccharides, or polyphosphazenes.

Suitable Th1 adjuvant systems that may be used include, Monophosphoryllipid A, particularly 3-de-O-acylated monophosphoryl lipid A, and acombination of monophosphoryl lipid A, preferably 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) together with an aluminium salt(preferably aluminium phosphate). An enhanced system involves thecombination of a monophosphoryl lipid A and a saponin derivativeparticularly the combination of QS21 and 3D-MPL as disclosed in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol as disclosed in WO96/33739. A particularly potentadjuvant formulation involving QS21 3D-MPL and tocopherol in an oil inwater emulsion is described in WO95/17210 and is a preferredformulation.

The vaccine may comprise a saponin, more preferably QS21. It may alsocomprise an oil in water emulsion and tocopherol. Unmethylated CpGcontaining oligo nucleotides (WO 96/02555) are also preferentialinducers of a TH1 response and are suitable for use in the presentinvention.

The vaccine preparation of the present invention may be used to protector treat a mammal susceptible to infection, by means of administeringsaid vaccine via systemic or mucosal route. These administrations mayinclude injection via the intramuscular, intraperitoneal, intradermal orsubcutaneous routes; or via mucosal administration to theoral/alimentary, respiratory, genitourinary tracts. Thus one aspect ofthe present invention is a method of immunizing a human host against adisease caused by infection of a gram-negative bacteria, which methodcomprises administering to the host an immunoprotective dose of the OMVpreparation of the present invention.

The amount of antigen in each vaccine dose is selected as an amountwhich induces an immunoprotective response without significant, adverseside effects in typical vaccinees. Such amount will vary depending uponwhich specific immunogen is employed and how it is presented. Generally,it is expected that each dose will comprise 1-100 μg of protein antigenor OMV preparation, preferably 5-50 μg, and most typically in the range5-25 μg.

An optimal amount for a particular vaccine can be ascertained bystandard studies involving observation of appropriate immune responsesin subjects. Following an initial vaccination, subjects may receive oneor several booster immunisations adequately spaced.

The vaccines of the invention are preferably immunoprotective andnon-toxic and suitable for paediatric or adolescent use.

By paediatric use it is meant use in infants less than 4 years old.

By immunoprotective it is meant that the SBA and/or animal protectionmodel and/or adhesion blocking assay described above are satisfactorilymet.

By non-toxic it is meant that there is no more than a satisfactory levelof endotoxin activity in the vaccine as measured by the well-known LALand pyrogenicity assays.

Polynucleotides of the Invention

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

Another aspect of the invention relates to an immunological/vaccineformulation which comprises one or more polynucleotide(s). Suchtechniques are known in the art, see for example Wolff et al., Science,(1990) 247: 1465-8.

Such vaccines comprise one or more polynucleotide(s) encoding aplurality of proteins corresponding to protein combinations of theinvention described above.

The expression of proteins from such polynucleotides would be under thecontrol of a eukaryotic promoter capable of driving expression within amammalian cell. The polynucleotide may additionally comprise sequenceencoding other antigens. Examples of eukaryotic promoters that coulddrive the expression include viral promoters from viruses includingadenoviral promoters, retroviral promoters. Alternatively, mammalianpromoters could be used to drive expression.

Further Aspects of the Invention

Another aspect of the invention involves a method for treatment orprevention of Neisserial disease comprising administering a protectivedose (or effective amount) of the vaccine of the invention to a host inneed thereof. Neisseria meningitidis serogroups A, B, C, Y or W135and/or Neisseria gonorrhoeae infection could be advantageously preventedor treated.

The invention also includes a use of the vaccine of the invention in thepreparation of a medicament for treatment of prevention of Neisserialinfection. Again Neisserial infection encompasses infection by Neisseriameningitidis serogroups A, B, C, Y, W-135 and/or Neisseria gonorrhoeae.

Another aspect of the invention is a genetically engineered Neisserialstrain from which an outer membrane vesicle of the inventions (having atleast two proteins of the invention recombinantly upregulated, asdescribed above) may be derived. Such Neisserial strains may beNeisseria meningitidis or Neisseria gonorrhoeae.

The strain may also have been engineered (as described above) todownregulate the expression of other Neisserial proteins including theexpression of one, two, three, four, five, six, seven or eight of LgtB,LgtE, SiaD, OpC, OpA, PorA, FrpB, msbB and HtrB. Preferred combinationsfor downregulation include down regulation (preferably deletion) of atleast LgtB and SiaD, downregulation of at least PorA and OpC,downregulation of at least PorA and OpA and downregulation of at leastPorA, OpA and OpC.

Further aspects of the invention are methods of making the immunogeniccomposition or vaccine of the invention. These include a methodcomprising a step of mixing together at least two isolated antigens orproteins from Neisseria, which may be present in the form of blebsderived from the Neisserial strains of the invention, to make animmunogenic composition of the invention, and a method of making thevaccine of the invention comprising a step of combining the immunogeniccomposition of the invention with a pharmaceutically acceptable carrier.

Also included in the invention are methods of making the immunogeniccomposition of the invention comprising a step of isolating outermembrane vesicles of the invention from a Neisserial culture. Such amethod may involve a further step of combining at least two outermembrane vesicle preparations, preferably wherein at least one outermembrane vesicle preparation contains LPS of immunotype L2 and at leastone outer membrane vesicle preparation contains LPS of immunotype L3.The invention also includes such methods wherein the outer membranevesicles are isolated by extracting with a concentration of DOC of0-0.5%. DOC concentrations of 0.3%-0.5% are used to minimise LPScontent. In OMV preparations where LPS is to be conserved as an antigen,DOC concentrations of 0-0.3%, preferably 0.05%-0.2%, most preferably ofabout 0.1% are used for extraction.

Ghost or Killed Whole Cell Vaccines

The inventors envisage that the above improvements to bleb preparationsand vaccines can be easily extended to ghost or killed whole cellpreparations and vaccines (with identical advantages). The modifiedGram-negative strains of the invention from which the bleb preparationsare made can also be used to made ghost and killed whole cellpreparations. Methods of making ghost preparations (empty cells withintact envelopes) from Gram-negative strains are well known in the art(see for example WO 92/01791). Methods of killing whole cells to makeinactivated cell preparations for use in vaccines are also well known.The terms ‘bleb [or OMV] preparations’ and ‘bleb [or OMV] vaccines’ aswell as the processes described throughout this document are thereforeapplicable to the terms ‘ghost preparation’ and ‘ghost vaccine’, and‘killed whole cell preparation’ and ‘killed whole cell vaccine’,respectively, for the purposes of this invention.

Antibodies and Passive Immunisation

Another aspect of the invention is a method of preparing an immuneglobulin for use in prevention or treatment of Neisserial infectioncomprising the steps of immunising a recipient with the vaccine of theinvention and isolating immune globulin from the recipient. An immuneglobulin prepared by this method is a further aspect of the invention. Apharmaceutical composition comprising the immune globulin of theinvention and a pharmaceutically acceptable carrier is a further aspectof the invention which could be used in the manufacture of a medicamentfor the treatment or prevention of Neisserial disease. A method fortreatment or prevention of Neisserial infection comprising a step ofadministering to a patient an effective amount of the pharmaceuticalpreparation of the invention is a further aspect of the invention.

Inocula for polyclonal antibody production are typically prepared bydispersing the antigenic composition in a physiologically tolerablediluent such as saline or other adjuvants suitable for human use to forman aqueous composition. An immunostimulatory amount of inoculum isadministered to a mammal and the inoculated mammal is then maintainedfor a time sufficient for the antigenic composition to induce protectiveantibodies.

The antibodies can be isolated to the extent desired by well knowntechniques such as affinity chromatography (Harlow and Lane Antibodies;a laboratory manual 1988).

Antibodies can include antiserum preparations from a variety of commonlyused animals e.g. goats, primates, donkeys, swine, horses, guinea pigs,rats or man. The animals are bled and serum recovered.

An immune globulin produced in accordance with the present invention caninclude whole antibodies, antibody fragments or subfragments. Antibodiescan be whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD orIgE, chimeric antibodies or hybrid antibodies with dual specificity totwo or more antigens of the invention. They may also be fragments e.g.F(ab′)2, Fab′, Fab, Fv and the like including hybrid fragments. Animmune globulin also includes natural, synthetic or geneticallyengineered proteins that act like an antibody by binding to specificantigens to form a complex.

A vaccine of the present invention can be administered to a recipientwho then acts as a source of immune globulin, produced in response tochallenge from the specific vaccine. A subject thus treated would donateplasma from which hyperimmune globulin would be obtained viaconventional plasma fractionation methodology. The hyperimmune globulinwould be administered to another subject in order to impart resistanceagainst or treat Neisserial infection. Hyperimmune globulins of theinvention are particularly useful for treatment or prevention ofNeisserial disease in infants, immune compromised individuals or wheretreatment is required and there is no time for the individual to produceantibodies in response to vaccination. An additional aspect of theinvention is a pharmaceutical composition comprising two of moremonoclonal antibodies (or fragments thereof; preferably human orhumanised) reactive against at least two constituents of the immunogeniccomposition of the invention, which could be used to treat or preventinfection by Gram negative bacteria, preferably Neisseria, morepreferably Neisseria meningitidis or Neisseria gonorrhoeae and mostpreferably Neisseria meningitidis serogroup B.

Such pharmaceutical compositions comprise monoclonal antibodies that canbe whole immunoglobulins of any class e.g. IgG, IgM, IgA, IgD or IgE,chimeric antibodies or hybrid antibodies with specificity to two or moreantigens of the invention. They may also be fragments e.g. F(ab′)2,Fab′, Fab, Fv and the like including hybrid fragments.

Methods of making monoclonal antibodies are well known in the art andcan include the fusion of splenocytes with myeloma cells (Kohler andMilstein 1975 Nature 256; 495; Antibodies—a laboratory manual Harlow andLane 1988). Alternatively, monoclonal Fv fragments can be obtained byscreening a suitable phage display library (Vaughan T J et al 1998Nature Biotechnology 16; 535). Monoclonal antibodies may be humanised orpart humanised by known methods.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

The terms “comprising”, “comprise” and “comprises” herein is intended bythe inventors to be optionally substitutable with the terms “consistingof”, “consist of”, and “consists of”, respectively, in every instance.

Method of Industrial Application of the Invention

The examples below are carried our using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Methods for Constructing Strains of Neisseria meningitidisSerogroup B Used in Outer Membrane Vesicle Preparations

WO01/09350 provides detailed methods for preparing outer membranevesicles and manipulating the bacterial strains from which the outermembrane vesicles are derived. Where the outer membrane vesicles are toretain lipoproteins such as TbpB and or lipopolysaccharides, methods ofisolation with low levels or no deoxycholate are preferred.

Example 2 Up-Regulation of the Hsf Protein Antigen in a RecombinantNeisseiria meningitidis Serogroup B Strain Lacking Functional Cps Genesbut Expressing PorA

As described in WO01/09350 examples, in certain countries, the presenceof PorA in outer membrane vesicles may be advantageous, and canstrengthen the vaccine efficacy of recombinant improved blebs. In thefollowing example, we have used a modified pCMK(+) vector to up-regulatethe expression of the Hsf protein antigen in a strain lacking functionalcps genes but expressing PorA. The original pCMK(+) vector contains achimeric porA/lacO promoter repressed in E. coli host expressinglacy^(q) but transcriptionally active in Neisseria meningitidis. In themodified pCMK(+), the native porA promoter was used to drive thetranscription of the hsf gene. The gene coding for Hsf was PCR amplifiedusing the HSF 01-NdeI and HSF O₂-NheI oligonucleotide primers, presentedin the table below. Because of the sequence of the HSF 01-NdeI primerthe Hsf protein expressed will contain two methionine residues at the 5′end. The conditions used for PCR amplification were those described bythe supplier (HiFi DNA polymerase, Boehringer Mannheim, GmbH). Thermalcycling was the following: 25 times (94° C. 1 min., 48° C. 1 min., 72°C. 3 min.) and 1 time (72° C. 10 min., 4° C. up to recovery). Thecorresponding amplicon was subsequently cloned in the correspondingrestriction sites of pCMK(+) delivery vector. In this recombinantplasmid, designed pCMK(+)-Hsf, we deleted the lacO present in thechimeric porA/lacO promoter by a recombinant PCR strategy. ThepCMK(+)-Hsf plasmid was used as a template to PCR amplify 2 separate DNAfragments:

-   -   fragment 1 contains the porA 5′ recombinogenic region, the        Kanamycin resistance gene and the porA promoter. Oligonucleotide        primers used, RP1 (SacII) and RP2, are presented in the table        below. RP1 primer is homologous to the sequence just upstream of        the lac operator.    -   fragment 2 contains the Shine-Dalgarno sequence from the porA        gene, the hsf gene and the porA 3′ recombinogenic region.        Oligonucleotide primers used, RP3 and RP4(ApaI), are presented        in the table below. RP3 primer is homologous to the sequence        just downstream of the lac operator. The 3′ end of fragment 1        and the 5′ end of fragment 2 have 48 bases overlapping. 500 ng        of each PCR (1 and 2) were used for a final PCR reaction using        primers RP1 and RP4. The final amplicon obtained was subcloned        in pSL1180 vector restricted with SacII and ApaI. The modified        plasmid pCMK(+)-Hsf was purified at a large scale using the        QIAGEN maxiprep kit and 2 μg of this material was used to        transform a Neisseiria meningitidis serogroup B strain lacking        functional cps genes. In order to preserve the expression of        porA, integration resulting from a single crossing-over was        selected by a combination of PCR and Western blot screening        procedures. Kanamycin resistant clones testing positive by        porA-specific PCR and western blot were stored at −70° C. as        glycerol stocks and used for further studies. Bacteria        (corresponding to about 5.10⁸ bacteria) were re-suspended in 50        μl of PAGE-SDS buffer, frozen (−20° C.)/boiled (100° C.) three        times and then were separated by PAGE-SDS electrophoresis on a        12.5% gel. The expression of Hsf was examined in Whole-cell        bacterial lysates (WCBL) derived from NmB [Cps−, PorA+] or NmB        [Cps−, PorA⁺, Hsf+]. Coomassie staining detected a significant        increase in the expression of Hsf (with respect to the        endogenous Hsf level). This result confirms that the modified        pCMK(+)-Hsf vector is functional and can be used successfully to        up-regulate the expression of outer membrane proteins, without        abolishing the production of the major PorA outer membrane        protein antigen.        Oligonucleotides Used in this Work

Oligonucleotides Sequence Remark(s) Hsf01-Nde5′-GGA ATT CCA TAT GAT GAA CAA NdeI cloning AAT ATA CCG C-3′ siteHsf02-Nhe 5′-GTA GCT AGC TAG CTT ACC ACT Nhe I cloning GAT AAC CGA C-3′site GFP-mut-Asn 5′-AAC TGC AGA ATT AAT ATG AAA AsnI cloningGGA GAA GAA CTT TTC-3′ site  Compatible with NdeI GFP-Spe5′-GAC ATA CTA GTT TAT TTG TAG SpeI cloning AGC TCA TCC ATG-3′ site Compatible with NheI RP1 (SacII) 5′-TCC CCG CGG GCC GTC TGA ATASacII cloning CAT CCC GTC-3′ site RP2 5′-CAT ATG GGC TTC CTT TTG TAAATT TGA GGG CAA ACA CCC GAT ACG TCT TCA-3′ RP35′-AGA CGT ATC GGG TGT TTG CCC TCA AAT TTA CAA AAG GAA GCC CAT ATG-3′RP4(ApaI) 5′-GGG TAT TCC GGG CCC TTC AGA ApaI cloning CGG CGC AGC AGG-3′site

Example 3 Up-regulation of the N. meningitidis serogroup B tbpA Gene byPromoter Replacement

The aim of the experiment was to replace the endogenous promoter regionof the tbpA gene by the strong porA promoter, in order to up-regulatethe production of the TbpA antigen. For that purpose, a promoterreplacement plasmid was constructed using E. coli cloning methodologies.A DNA region (731 bp) located upstream from the tbpA coding sequence wasdiscovered in the private Incyte PathoSeq data base of the Neisseriameningitidis strain ATCC 13090. This DNA contains the sequence codingfor TbpB antigen. The genes are organized in an operon. The tbpB genewill be deleted and replaced by the CmR/porA promoter cassette. For thatpurpose, a DNA fragment of 3218 bp corresponding to the 509 bp 5′flanking region of tbpB gene, the 2139 bp tbpB coding sequence, the 87bp intergenic sequence and the 483 first nucleotides of tbpA codingsequence was PCR amplified from Neisseria meningitidis serogroup Bgenomic DNA using oligonucleotides BAD16 (5′-GGC CTA GCT AGC CGT CTG AAGCGA TTA GAG TTT CAA AAT TTA TTC-3′) and BAD17 (5′-GGC CAA GCT TCA GACGGC GTT CGA CCG AGT TTG AGC CTT TGC-3′) containing uptake sequences andNheI and HindIII restriction sites (underlined). This PCR fragment wascleaned with a High Pure Kit (Boerhinger Mannheim, Germany) and directlycloned in a pGemT vector (Promega, USA). This plasmid was submitted tocircle PCR mutagenesis (Jones & Winistofer (1992)) in order to (i)insert suitable restriction sites allowing cloning of a CmR/PorApromoter cassette and (ii) to delete 209 bp of the 5′ flanking sequenceof tbpB and the tbpB coding sequence. The circle PCR was performed usingthe BAD 18 (5′-TCC CCC GGG AAG ATC TGG ACG AAA AAT CTC AAG AAA CCG-3′) &the BAD 19 (5′-GGA AGA TCT CCG CTC GAG CAA ATT TAC AAA AGG AAG CCG ATATGC AAC AGC AAC ATT TGT TCC G-3′) oligonucleotides containing suitablerestriction sites Xmal, BglII and XhoI (underlined). The CmR/PorApromoter cassette was amplified from the pUC D15/Omp85 plasmidpreviously described, using primers BAD21 (5′-GGA AGA TCT CCG CTC GAGACA TCG GGC AAA CAC CCG-3′) & BAD20 (5′-TCC CCC GGG AGA TCT CAC TAG TATTAC CCT GTT ATC CC-3′) containing suitable restriction sites Xmal, SpeI,BglII and XhoI (underlined). This PCR fragment was cloned in the circlePCR plasmid. This plasmid will be used to transform Neisseriameningitidis serogroup B □cps-□ and □cps- porA-□ strains. Integration bydouble crossing-over in the upstream region of tbpA will direct theinsertion of the porA promoter directly upstream of the tbpA ATG.

Example 4 Construction of a N. meningitidis Serogroup B StrainUp-Regulated for the Expression of Two Antigens: TbpA and Hsf.

The aim of the experiment was to up-regulate the expression of TbpA andHsf simultaneously in the same N. meningitidis serogroup B strain. Theproduction of TbpA was up-regulated by replacing its endogenous promoterregion by the strong porA promoter (promoter replacement). In thiscontext, the tbpB gene, located upstream of tbpA is deleted, and theTbpB protein no longer present in the outer-membrane. The expression ofHsf was up-regulated by insertion (homologous recombination) of a secondcopy of the corresponding gene at the porA locus (gene delivery). Bothstrains have been described in a separate patent referred to asWO01/09350. The selection markers used in both strategies (Cm^(R) orKan^(R)) allowed the combination of both integrations into the samechromosome.

Total genomic DNA was extracted from the recombinant Nm.Bcps−/TbpA+/PorA+strain by the Qiagen Genomic tip 500-G protocol. Ten ngof DNA was restricted o/n with DraIII restriction enzyme and used totransform Neisseria meningitidis serogroup B by the classicaltransformation protocol. Cells used for transformation were eitherrecombinant NmB cps−/Hsf+/PorA+(homologous recombination by 1 crossingover into the porA locus) or recombinant NmB cps−/Hsf+/PorA− (Allelicexchange/homologous recombination by 2 crossing over into the porAlocus). They were plated over-night on GC agar containing 200 μg/mlkanamycine, diluted to DO₆₅₀=0.1 in GC liquid medium 10 mM MgCl₂, andincubated 6 hours at 37° C. under vigorous agitation with 10 μg ofDraIII restricted genomic DNA. Recombinant Neisseria meningitidisresulting from a double crossing over event (PCR screening) wereselected on GC medium containing 200 μg/ml kanamycin and 5 μg/mlchloramphenicol and analyzed for TbpA and Hsf expression in OMVpreparations. As represented in FIG. 1, the production of both TbpA andHsf was significantly increased in the OMV prepared from the TbpA/Hsfrecombinant NmB strain when compared to the OMV prepared from thecontrol NmB cps−strains. The level of over expression of each protein inthe dual recombinant is comparable with the level of expression obtainedin the corresponding single recombinants. The level of over expressionof TbpA and Hsf was comparable in PorA+ and PorA− strains (data notshown). All together, these data demonstrate that: (i) expression ofTbpA and Hsf can be jointly and concomitantly up-regulated into N.meningitidis and (ii) recombinant blebs enriched for TbpA and Hsf can beobtained and used for immunization.

Example 5 Construction of a N. meningitidis Serogroup B StrainUp-Regulated for the Expression of Two Antigens: TbpA and NspA

The aim of the experiment was to up-regulate the expression of TbpA andNspA simultaneously in the same N. meningitidis serogroup B strain. Theproduction of TbpA was up-regulated by replacing its endogenous promoterregion by the strong porA promoter (promoter replacement). Theexpression of NspA was up-regulated by insertion (homologousrecombination) of a second copy of the corresponding gene at the porAlocus (gene delivery). Both individual strains have been described in aseparate patent WO01/09350. The selection markers used in bothstrategies (Cm^(R) or Kan^(R)) allowed the combination of bothintegrations into the same chromosome. Total genomic DNA was extractedfrom the recombinant NmB cps−/TbpA+/PorA+strain by the Qiagen Genomictip 500-G protocol. Ten μg of DNA was restricted o/n with AatIIrestriction enzyme and used to transform Neisseria meningitidisseregroup B by the classical transformation protocol. Cells used fortransformation were recombinant NmB cps−/NspA+/PorA−. They were platedover-night on GC agar containing 200 μg/ml kanamycine, diluted toDO₆₅₀=0.1 in GC liquid medium 10 mM MgCl₂, and incubated 6 hours at 37°C. under vigorous agitation with 10 μg of AatII restricted genomic DNA.Recombinant Neisseria meningitidis resulting from a double crossing overevent (PCR screening) were selected on GC medium containing 200 μg/mlkanamycine and 5 μg/ml chloramphenicol and analyzed for TbpA and NspAexpression in OMV preparations. The production of both TbpA and NspA wassignificantly increased in the OMV prepared from the TbpA/NspArecombinant NmB strain when compared to the OMV prepared from thecontrol NmB cps−strains. The level of over-expression of each protein inthe dual recombinant is comparable with the level of expression obtainedin the corresponding single recombinants. All together, these datademonstrate that: (i) expression of TbpA and NspA can be jointly andconcomitantly up-regulated into N. meningitidis and (ii) recombinantblebs enriched for TbpA and NspA can be obtained and used forimmunization.

Example 6 Construction of a N. meningitidis Serogroup B StrainUp-Regulated for the Expression of Two Antigens: NspA and D15/Omp85

The aim of the experiment was to up-regulate the expression of NspA andD15/Omp85 simultaneously in the same N. meningitidis serogroup B strain.The production of D15/Omp85 was up-regulated by replacing its endogenouspromoter region by the strong porA promoter (promoter replacement). Theexpression of NspA was up-regulated by insertion (homologousrecombination) of a second copy of the corresponding gene at the porAlocus (gene delivery). Both strains have been described in a separatepatent WO01/09350. The selection markers used in both strategies (Cm^(R)or Kan^(R)) allowed the combination of both integrations into the samechromosome.

Total genomic DNA was extracted from the recombinant NmBcps−/D15−Omp85/PorA+strain by the Qiagen Genomic tip 500-G protocol. Tenμg of DNA was restricted o/n with AatII restriction enzyme and used totransform Neisseria meningitidis seregroup B by the classicaltransformation protocol. Cells used for transformation were recombinantNmB cps−/NspA+/PorA−. They were plated o/n on GC agar containing 200μg/ml kanamycine, diluted to DO₆₅₀=0.1 in GC liquid medium 10 mM MgCl₂,and incubated 6 hours at 37° C. under vigorous agitation with 10 μg ofAatII restricted genomic DNA. Recombinant Neisseria meningitidisresulting from a double crossing over event (PCR screening) wereselected on GC medium containing 200 μg/ml kanamycine and 5 μg/mlchloramphenicol and analyzed for NspA and D15/Omp85 expression in OMVpreparations. The production of both NspA and D15/Omp85 wassignificantly increased in the OMV prepared from the NspA/D15−Omp85recombinant NmB strain when compared to the OMV prepared from thecontrol NmB cps−strains. The level of over expression of each proteinsin the dual recombinant is comparable with the level of expressionobtained in the corresponding single recombinants. All together, thesedata demonstrate that: (i) expression of NspA and Omp85 can be jointlyand concomitantly up-regulated into N. meningitidis and (ii) recombinantblebs enriched for NspA and Omp85 can be obtained and used forimmunization.

Example 7 Production and Purification of Recombinant Hsf Forms in E.coli

Computer analysis of the Hsf-like protein from Neisseria meningitidisreveals at least four structural domains. Considering the Hsf sequencefrom strain H44/76 as a reference, Domain 1, comprising amino-acid 1 to51, encodes a sec-dependant signal peptide characteristic of theauto-transporter family, Domain 2, comprising amino-acids 52 to 473,encode the passenger domain likely to be surface exposed and accessibleto the immune system, Domain 3, comprising amino-acids 474 to 534,encodes a putative coiled-coil domain required for proteinoligomerisation and a hinge (neck), Domain 4, comprising residues 535 tothe C-terminus, is predicted to encode a beta-strands likely to assembleinto a barrel-like structure and to be anchored into the outer-membrane(Henderson et al. (1998), Trends Microbiol. 6: 370-378; Hoiczyk et al.(2000), EMBO 22: 5989-5999). Since domains 2 and 3 are likely to besurface-exposed, are well conserved (more than 80% in all strain tested;as described in Pizza et al. (2000), Science 287: 1816-1820), theyrepresent interesting vaccine candidates. For that purpose, domain 2(referred to as Hsf passenger domain) and domain 2+3 (referred to as Hsfneck+coiled-coil domain) were expressed in and purified from E. coli.DNA fragments encoding amino-acids 52-473 (Hsf passenger) and 52-534(Hsf n+cc) were PCR amplified using oligonucleotides adding terminalReal (forward primer) and XhoI (reverse primer) restriction sites.Purified amplicons were digested with RcaI/XhoI in the conditionsrecommended by the supplier, and were subsequently cloned into the NcoI(compatible with real)/XhoI sites of the pET24d (Novagen Inc., MadisonWis.) E. coli expression vector. Recombinant plasmids were selected andused to prepare purified recombinant plasmids. For expression study,these vectors (pET-Hsf pas & pET-Hsf ncc) were introduced into theEscherichia coli strain B121DE3 (Novagen), in which, the gene for the T7polymerase is placed under the control of the isopropyl-beta-Dthiogalactoside (IPTG)-regulatable lac promoter. Liquid cultures (700ml) of the Novablue (DE3) [pET-24b/BASB029] E. coli recombinant strainwere grown at 37° C. under agitation until the optical density at 600 nm(OD₆₀₀) reached 0.6. At that time-point, IPTG was added at a finalconcentration of 1 mM and the culture was grown for 4 additional hours.The culture was then centrifuged at 10,000 rpm and the pellet was frozenat −20° C. for at least 10 hours. After thawing, the pellet (680 mlculture) was resuspended during 30 minutes at 22° C. in 20 mM phosphatebuffer pH 7.0 prior cell lysis by two passes through a Rannie disruptor.Lysed cells were pelleted 30 min at 15,000 rpm (Beckman J2-HScentrifuge, JA-20 rotor) at 4° C. The supernatant was loaded on aQ-Sepharose fast flow column (Pharmacia) equilibrated in 20 mM Tris-HClbuffer ph 8.0. After passage of the flowthrough, the column was washedwith 5 column volumes of 20 mM Tris-HCl buffer pH 8.0. The recombinantprotein was eluted from the column by 250 mM NaCl in 20 mM Tris-HClbuffer pH 8.0. Antigen positive fractions were pooled and dialyzedovernight against 20 mM phosphate buffer pH 7.0. 0.5M NaCl and 20 mMImidazole were added to the dialyzed sample. Sample was then appliedonto Ni-NTA Agarose column (Qiagen) equilibrated in 20 mM phosphatebuffer pH 7.0 containing 500 mM NaCl and 20 mM Imidazole. After passageof the flowthrough, the column was washed with 5 column volumes of 20 mMphosphate buffer pH 7.0 containing 500 mM NaCl and 20 mM Imidazole.Contaminants were eluted by 100 mM Imidazole in 20 mM phosphate bufferpH 7.0. The recombinant protein was eluted from the column by 250 mMImidazole in 20 mM phosphate buffer pH 7.0. Antigen positive fractionswere pooled and dialyzed versus 10 mM phosphate buffer pH 6.8 containing150 mM NaCl. As shown in FIG. 2, an enriched (purity estimated to morethan 90% pure in CBB stained SDS-PAGE) Hsf-like passenger protein,migrating at around 47 kDa (estimated relative molecular mass), waseluted from the column. This polypeptide was reactive against a mousemonoclonal antibody raised against the 5-histidine motif. Takentogether, these data indicate that the both Hsf passenger and Hsf nccgene can be expressed and purified under a recombinant form in E. coli.

Example 8 Production and Purification of Recombinant Hap Passenger in E.coli

Computer analysis of the Hap-like protein from Neisseria meningitidisreveals at least three structural domains. Considering the Hap-likesequence from strain H44/76 as a reference, Domain 1, comprisingamino-acid 1 to 42, encodes a sec-dependant signal peptidecharacteristic of the auto-transporter family, Domain 2, comprisingamino-acids 43 to 950, encode the passenger domain likely to be surfaceexposed and accessible to the immune system, Domain 3, comprisingresidues 951 to the C-terminus (1457), is predicted to encode abeta-strands likely to assemble into a barrel-like structure and to beanchored into the outer-membrane. Since domains 2 is likely to to besurface-exposed, well conserved (more than 80% in all strain tested) andcould be produced as subunit antigens in E. coli, it represents aninteresting vaccine candidates. Since domains 2 and 3 are likely to besurface-exposed, are well conserved (more than 80% in all strain tested;as described in Pizza et al. (2000), Science 287: 1816-1820), theyrepresent interesting vaccine candidates. For that purpose, domain 2(referred to as Hap passenger domain was expressed in and purified fromE. coli. A DNA fragment encoding amino-acids 43-950 (Hap passenger) wasPCR amplified using oligonucleotides adding terminal NcoI (forwardprimer) and XhoI (reverse primer) restriction sites. Purified ampliconswere digested with NcoI/XhaI in the conditions recommended by thesupplier, and were subsequently cloned into the NcoI/XhoI sites of thepET24d (Novagen Inc., Madison Wis.) E. coli expression vector.Recombinant plasmids were selected and purified to large scale. Forexpression study, these vectors (pET-Hap pass) were introduced into theEscherichia coli strain B121DE3 (Novagen), in which, the gene for the T7polymerase is placed under the control of the isopropyl-beta-Dthiogalactoside (IPTG)-regulatable lac promoter.

Cultivation of E. coli BL21 [pET-Hau pass] in fermentor: An aliquotefraction (100 μl) from the master seed was spread on FEC013AA plates(Soja peptone A3 20 g/L, yeast extract 5 g/L, NaC1 5 g/L, Agar 18 g/L,distillated H₂O up to 1 L) and grown 20 hours at 37° C. The bacteriallawn was harvested and resuspended in sterile water containing NaC10.9%. This solution was used to inoculate a 20 L fermentor used in thebatch mode in FEC011AC medium (Soja peptone 24 g/L, Yeast extract 48g/L, MgSO4/7H2O 0.5 g/L, K2HPO4 2 g/L, NaH2PO4/2H2O 0.45 g/L, Glycerol(87%) 40 g and distilated H₂O up to 1 L). Temperature (30° C.), pH (6.8,NaOH 25%/H₃PO₄ 25%), pressure (500 mbar), were maintained constant andaeration was set to 20 L/min. In these conditions dissolved oxygenpressure was maitained to 20% by tuning agitation (100 to 1000 rpm).Inducer (IPTG, 1 mM) was added after 8 hours of growth (OD=27.8).Samples (6 L) were collected after 6 hours (OD=49.2) and 16H30(OD=48.6), biomass was harvested by centrifugation and correspondingpellets stored at −20° C.

Purification of Hap Passenger:

HAP passenger was purified from a fermentor in batch mode. Apurification scheme was developed (see below).

The majority of Hap passenger is recovered in the centrifugation pelletafter cell breakage. Solubilization was made possible by 8M urea.Despite N-term His-tail, IMAC was not operative as 1st step, but wellafter a first step on SP-XL cation-exchanger. On this SP-Sepharose-XL,the protein is eluted quantitatively in the middle of a linear NaCl(0-250 mM) gradient. IMAC was done with Cu⁺⁺-loaded Chelating SepharoseFF, as for FHAb. This time, contrary to FHAb, IMAC shows a significantpurification factor. On SDS-PAGE, HAP⅔ seems pure after IMAC. The HAP⅔peak was however very broad on 0-200 mM imidazole gradient, so we triedelution by imidazole steps (10 mM-100 mM); gradient mode seems howevermore efficient in terms of purity. As final step, we tried theurea-to-arginine buffer exchange by gel permeation however in this casethe protein eluted on two peaks. These two peaks show a comparableprofile on SDS-PAGE; so it can be hypothetized that it is due to apartial refolding of HAP passenger. We then went back to the classicaldialysis as final step for buffer exchange. SDS-PAGE analysis shows goodpurity of the final material (see in FIG. 3). HAP passneger purity isfurther confirmed by WB anti-his. It is recognized by anti-E. coli. AMolecular weight of 96.1 KD is found.

Example 9 Production and Purification of Recombinant FrpA/C Forms in E.coli

Neisseria meningitidis encodes two RTX proteins, referred to as FrpA &FrpC secreted upon iron limitation (Thompson et al., (1993) J.Bacteriol. 175:811-818; Thompson et al., (1993) Infect. Immun61:2906-2911). The RTX (Repeat ToXin) protein family have in common aseries of 9 amino acid repeat near their C-termini with the consensus:Leu Xaa Gly Gly Xaa Gly (Asn/Asp) Asp Xaa. (LXGGXGN/_(D)DX). The repeatsin E. coli H1yA are thought to be the site of Ca2+ binding. Asrepresented in FIG. 4, meningococcal FrpA and FrpC proteins, ascharacterized in strain FAM20, share extensive amino-acid similarity intheir central and C-terminal regions but very limited similarity (ifany) at the N-terminus Moreover, the region conserved between FrpA andFrpC exhibit some polymorphism due to repetition (13 times in FrpA and43 times in FrpC) of a 9 amino acid motif To evaluate the vaccinepotential of FrpA & FrpC, we produced recombinantly in E. coli proteinregions conserved between FrpA and FrpC. For that purpose, a DNA segmentcovering aminoacids 277 to 1007 (with regard to the N. meningitidisFAM20 peptide sequence) was PCR amplified from the N. meningitidisserogroupB H44/76 genome using forward primers FrpA-19(5′-CTCGAGACCATGGGCAAA TATCATGTCTACGACCCCCTCGC-3′) and reverse primerFrpA-18 (3′-GTG CATAGTGTCAGAGTTTTTGTCGACGTCGTAATTATAGACC-3′). Threeamplicons of respectively ˜1530 bp (3 repeats), ˜2130 bp (13 repeats)and 2732 bp (23 repeats) were obtained and digested with NcoI and SalIrestriction endonucleases. These fragments were then inserted into theNcoIIXhoI (compatible with SalI) sites of pET24d and recombinantplasmids (pET-Frp3, pET-Frp13 and pET-Frp23 respectively) were selectedand used to transform E. coli BL21DE3 cells. As represented in FIG. 5,all three constructs produced recombinant FrpA/C conserved domains uponinduction. Moreover, increasing the number of repeats increased thesolubility of the recombinant protein, as determined by cellfractionation analysis (data not shown).

Purification of FrpA/C conserved domain containing 23 repeats of thenonapeptide LXGGXGN/DDX (911 aa in total): 3.5 liters of E. coliB121DE3[pET-Frp23] were cultivated and induced for 4 hours by additionof 2 mM IPTG when OD reached 0.6. Cell were harvested by centrifugationand the correspondeing pellet was pressure-disrupted, clarified bycentrifugation and the corresponding supernatent loaded on a Ni2+-ionmetal affinity column (Ni-NTA-agarose, Qiagen GmBh). Imidazole ( ) wasused for elution and was finally removed by the extensive dialysisagainst 10 mM Na phosphate pH6.8, 150 mM NaCl.

Example 10 Production and Purification of Recombinant FHA Forms in E.coli

Cloning of a truncated FhaB from N. meningitidis

Genomic DNA was extracted from 10¹⁰ cells of N. meningitidis serogroup Bstrain H44/76 using the QIAGEN genomic DNA extraction kit (Qiagen Gmbh).This material (1 μg) was then submitted to Polymerase Chain Reaction DNAamplification using the following primers specific of the FhaB gene:JKP: 5′AAT GGA ATA CAT ATG AAT AAA GGT TTA CAT CGC ATT ATC3′ and 57JKP5′CCA ACT AGT GTT TTT CGC TAC TTG GAG CTG T3′. A DNA fragment of about4200 bp, encoding the first 1433 N-terminal amino acids of the protein,was obtained, digested by the NdeI/SpeI restriction endonucleases andinserted into the corresponding sites of the pMG MCS (pMG derivative,Proc Natl Acad Sci USA 1985 January; 82(1):88-92) using standardmolecular biology techniques (Molecular Cloning, a Laboratory Manual,Second Edition, Eds: Sambrook, Fritsch & Maniatis, Cold Spring Harborpress 1989)). The DNA sequence of the cloned FhaB fragment wasdetermined using the Big Dye Cycle Sequencing kit (Perkin-Elmer) and anABI 373A/PRISM DNA sequencer (see FIG. 1). The recombinant pMG-FhaBplasmid (lng) was then submitted to Polymerase Chain Reaction DNAamplification using primers specific FhaB (XJKP035′AATGGAATACATATGAATAAAGGTTTACATCGCATTATCTTTAG3′ and XJKP57025′GGGGCCACTCGAGGTTTTTCGCTACTTGGAGCTGTTTCAG ATAGG3′). A 4214 bp DNAfragment was obtained, digested by the NdeI/XhoI restrictionendonucleases and inserted into the corresponding sites of the pET-24bcloning/expression vector (Novagen) using standard molecular biologytechniques (Molecular Cloning, a Laboratory Manual, Second Edition, Eds:Sambrook, Fritsch & Maniatis, Cold Spring Harbor press 1989).Confirmatory sequencing of the recombinant pET-24b containing thetruncated FhaB (pET24b/FhaB⅔) was performed using the Big Dyes kit(Applied biosystems) and analysis on a ABI 373/A DNA sequencer in theconditions described by the supplier. The resulting nucleotide sequenceis presentedin FIG. 6.

Expression and Purification of Recombinant Truncated Fhab Protein inEscherichia coli.

The construction of the pET24b/FhaB⅔ cloning/expression vector wasdescribed above. This vector harbours the truncated FhaB gene isolatedfrom the strain H44/76 in fusion with a stretch of 6 Histidine residues(at the C-terminus of the recombinant product), placed under the controlof the strong bacteriophage T7 gene 10 promoter. For expression study,this vector was introduced into the Escherichia coli strain Novablue(DE3) (Novagen), in which, the gene for the T7 polymerase is placedunder the control of the isopropyl-beta-D thiogalactoside(IPTG)-regulatable lac promoter. Liquid cultures (100 ml) of theNovablue (DE3) [pET24b/FhaB⅔] E. coli recombinant strain were grown at37° C. under agitation until the optical density at 600 nm (OD₆₀₀)reached 0.6. At that time-point, IPTG was added at a final concentrationof 1 mM and the culture was grown for 4 additional hours. The culturewas then centrifuged at 10,000 rpm and the pellet was frozen at −20° C.for at least 10 hours. After thawing, the pellet was resuspended during30 min at 25° C. in buffer A (6M guanidine hydrochloride, 0.1M NaH2PO4,0.01M Tris, pH 8.0), passed three-times through a needle and clarifiedby centrifugation (20000 rpm, 15 min). The sample was then loaded at aflow-rate of 1 ml/min on a Ni2+-loaded Hitrap column (PharmaciaBiotech). After passsage of the flowthrough, the column was washedsuccesively with 40 ml of buffer B (8M Urea, 0.1MNaH2PO4, 0.01M Tris, pH8.0), 40 ml of buffer C (8M Urea, 0.1MNaH2PO4, 0.01M Tris, pH 6.3). Therecombinant protein FhaB⅔/His6 was then eluted from the column with 30ml of buffer D (8M Urea, 0.1MNaH2PO4, 0.01M Tris, pH 6.3) containing 500mM of imidazole and 3 ml-size fractions were collected. As presented inFIG. 7, a highly enriched FhaB-⅔/His6 protein, migrating at around 154kDa (estimated relative molecular mass), was eluted from the column.This polypeptide was reactive against a mouse monoclonal antibody raisedagainst the 5-histidine motif. Taken together, these data indicate thatthe FhaB⅔ can be expressed and purified under a recombinant form in E.coli.

Immunization of Mice with Recombinant FhaB⅔/His

Partially purified recombinant FhaB⅔/His6 protein expressed in E. coliwas injected three times in Balb/C mice on days 0, 14 and 29 (10animals/group). Animals were injected by the subcutaneous route witharound 5 μg of antigen in two different formulations: either adsorbed on100 μg AlPO₄ or formulated in SBAS2 emulsion (SB62 emulsion containing 5μg MPL and 5 μg QS21 per dose). A negative control group consisting ofmice immunized with the SBAS2 emulsion only has also been added in theexperiment. Mice were bled on days 29 (15 days Post II) and 35 (6 daysPost III) in order to detect specific anti-FhaB antibodies. Specificanti-FhaB antibodies were measured on pooled sera (from 10 mice/group)by ELISA on purified recombinant FhaB⅔/His.

Example 11 Adhesion Blocking Activities of Mouse and Rabbit Sera RaisedAgainst FHA, Hap and Hsf Antigens

Proteins homologous to the meningococcal FHAB-like, Hsf-like andHap-like have been described previously to be important virulencedeterminant and to mediate bacterial adhesion of Bordetella pertussis(FHA) and Haemophilus influenzae (Hap and Hsf). Adhesion to epithelialand endothelial cells is known to be crucial for colonization of thenasopharynges and crossing of the blood-brain barrier by themeningococcus. Thus interfering with the adhesion of N. meningitidisrepresent a valuable approach to controle meningococcal colonization andinfection. Here we tested if anti-sera directed against themeningococcal FHAB ⅔^(rd), Hap-like and Hsf-like antigens were able tointerfer the adhesion of Neisseria meningitidis to endothelial cells.The following experimental procedure was used:

Inhibition of adhesion to HUVEC's: the meningococcal test strain used inthis study was a non-capsulated, non-piliated, Opa- and Opc-derivativeof strain NmA8013. Meningococcal cells (2.10E5 colony forming units(CFU) of the NmA8013 derivative) were incubated during 30 minutes at 37°C. in a medium composed of 400 μl of RPMI, 50 μl of fetale bovine serumand 50 μl of the serum to be tested for adhesion blocking properties.This mixture was then placed in a well containing confluent monolayersof human umbilical vein endothelial cells (HUVEC's) whose culture mediumhas been previously removed. Bacteria and HUVEC's cells were incubatedduring 4 hours at 37° C., under 5% CO2. Cell monolayers were then washedthree times with fresh RPMI serum and subsequently scrapped off theplate. CFU associated to HUVEC's cells was then determined serialdilution and plating of the cell lysate onto GC plates. Plates wereincubated during 48 hours at 37° C. to allow the recovery and growth ofcell-associated meningococci.

Adhesion-blocking activities of mouse and rabbit sera raised againstrecombinant FHAB⅔^(rd), Hap & hsf antigens: anti-FHA ⅔, anti-Hsffull-length (described in WO99/58683) and anti-Hap full-length(WO99/55873) antibodies, as well as anti-sera directed againstcorresponding Hsf & Hap passenger domains, interfere with meningococcaladhesion to endothelial HUVEC's cells. FIG. 8 illustrates that specificantibodies induced by FHA ⅔ formulated in AlPO₄ was able to inhibitNeisseria meningitidis B adhesion to the HUVEC cells compared to theadjuvant only. When compared to the SBAS2 adjuvant only (withoutantigen, group 4), the anti-FHA ⅔ abs (SBAS2 formulation) is stilleffective, but less potent than AlPO₄. The SBAS2 adjuvant only (withoutantigen) does not induce antibodies able to interfere with the adhesion.Compared to group 4, anti-Hap antibodies (group 1) may have a slightinhibition effect. In group 5, when a mixture of anti-FHA ⅔, anti-Hsfand anti-Hap antibodies is tested, inhibition of the adhesion isstronger than with anti-FHA ⅔ only, suggesting a synergetic effect givenby anti-Hap and anti-Hsf antibodies. In a second inhibition experiment(FIG. 2), a specific rabbit antiserum directed against anti OMVsover-expressing Hsf (as a candidate protein) was able to inhibitpartially the fixation of Neisseria meningitidis B to the endothelialcells compared to the negative control (group 3 vs 4). This rabbitantiserum has been demonstrated to contain a very high specific anti-Hsfantibody titer. Antibodies against rec Hsf (Hsf passenger and Hsf fulllength) are also able to inhibit adhesion of bacteria on the HUVECcells. This is true both with mice sera (groups 5-6) as well as withrabbit sera (in a laser extend) (groups 7-8). In this second experiment,specific anti-rec FHA ⅔ antibodies (group 1) already tested in the firstexperiment confirm their very high inhibitory effect. These resultsindicate that these specific antigens (Hap, FHA⅔ and Hsf), isolated orin combination, are interesting vaccine antigens.

Example 12 Protective Effect of Recombinant OMV's in the Mouse ChallengeModel

Several recombinant OMVs have been evaluated in Balb/C mice for theirprotective effect after lethal challenge. This active immunisation modelinvolved intraperitoneal injection of meningococci from several strains(suspended in iron depleted TSB medium) into adult Balb/C or OF1 mice(6-8 weeks old), after a series of immunization by the subcutaneousroute. The iron dextran, used as an external iron source seems to beneeded to maintain bacteraemia and induce mortality in infected animal.Although this IP model in mice has been shown to be effective forassessing virulence, immune protection and the role of iron ininfection, they do not incorporate the pharyngeal carriage phase, whichprecedes bacteraemia and meningitis in humans. This model has been usedto screen our several OMV candidates over-expressing NspA, TbpA, or Hsf.In the following experiments, Balb/C (inbred) or OF 1 (outbred) micewere immunized three times on days 0, 14 and 28 by the subcutaneousroute with 3 (PV00N049) to 5 μg (PV00N035 and PV00N043 experiments) ofrec. OMV over-expressing Hsf, NspA or TbpA formulated on Al(OH)₃ (100 μgAl(OH)₃/animal) (PV00N035 and PV00N043) or on Al PO₄ (100 μg AlPO₄/animal). Then, animals are bled on days 28 (day 14 past II) and 35(day 7 past III) for specific Ab evaluation. On day 35, 10 mg of irondextran are injected intraperitaneally one hour before the challenge.The challenges were done with H44/76 (B:15:P1.7, 16) or CU-385(B:4:P1.19, 15) strains, with around 1.10 e7 CFU/animal (see the tableof results for the exact challenge doses). The heterologous strain donewith the CU-385 strain is more stringent than when using the homologousstrain. Mortalities were recorded from days 1 to 5. The table 1hereafter illustrates that when compared to OMV porA (−) and with OMVporA (+) in a lesser extend, there is already a better protectionobserved with OMV TbpA (+) ( 1/10 and ⅗ for porA(−) and 9/10 and ⅗ forporA (+)), with OMV NspA (+) ( 4/10 and ⅘) and with OMV Hsf (+) ( 3/10,2/10 and ⅗). This is the global observation we can make in these threeexperiments. These data support that TbpA, Hsf and NspA antigens,expressed at the bleb surface, are of interest for a future menBvaccine.

TABLE 1 Protective activity in the mouse model of recombinantouter-membrane vesicles. The table summarizes the results obtainedduring three experiments (PV00N35, PV00N043 & PV00N049) OMVs (blebs)Survival rate (on day Active mouse protection PV00N035 PV00N043 PV00N049in OF1 mice in Balb/C mice in OF1 mice Immuno Challenge strain (+Specific Abs Rec OMVs H44/76 H44/76 CU-385 by Elisa (H44/76 background =B:15:P1.7, (B:15:P1.7, B:4:P1.19, Mean- porA P1.17,16) 1.27 1.0 11.1PV00N049 only OMV porA (−) 1/10 0/10 1/5 / OMV porA (+) 2/10 9/10 4/5 /OMV TbpA porA (+) NT 9/10 3/5 < OMV TbpA porA (−) NT 1/10 3/5 < OMV NspAporA (−) 1/10 4/10 4/5 155-(<   OMV Hsf PorA (−) 3/10 2/10 3/57802-(5496) OMV Hsf PorA (+) NT 9/9  NT / No antigen 0/10 0/10 1/5 / NT:Not tested.

Example 13 Protective Effect of Recombinant Subunit Antigens in theMouse Challenge Model

Several recombinant purified proteins have been evaluated in Balb/C micefor their protective effect after lethal challenge. This activeimmunization model involved introperitoneal injection of meningococcifrom several strains (suspended in iron depleted TSB medium) into adultBalb/C or OF 1 mice (6-8 weeks old) after a series of immunization bythe subcutaneous route.

The iron dextran, used as an external iron source seems to be needed tomaintain bacteraemia and induce mortality in infected animal. Althoughthis IP model in mice has been shown to be effective for assessingvirulence, immune protection and the role of iron in infection, they donot incorporate the pharyngeal carriage phase, which precedesbacteraemia and meningitis in humans. This model has been used to screenseveral menB sub-unit vaccine candidates like recombinant FrpC, TbpA,FHA⅔ and Hap molecules.

In this experiment, OF 1 (outbred) mice were immunized three times ondays 0, 14 and 28 by the subcutaneous route with 5 μg (PV00N050) ofthese proteins formulated on Al PO₄ (100 μg) in presence of 10 μg MPL(per animal). Then, animals are bled on days 28 (day 14 past II) and 35(day 7 past III) for specific Ab evaluation, while they are challengedon day 35. The day of challenge, 10 mg of iron dextran are injectedintraperitaneally one hour before the challenge. The challenges weredone with CU-385 strains (B:4:P1.19, 15), which is heterologous in thiscase, indeed, the antigens sequence coming from the H44/76 (B:15:P1.7,16), except for the TbpA for which the sequence comes from the B16B6strain (B:2a:P1.2).

The results illustrated in table 2 indicate that FrpC, TbpA, FHAB⅔^(rd),Hap induced significant protection in this model: from 2 to 4 out of 5mice survived after challenge, compared to only 1/5 with the adjuvantonly. In all groups but one, the specific antibody titer were high(specific anti-TbpA titer was moderate). All these data support thatFrpC, FrpA, FrpA/C conserved domain, TbpA, FHAB⅔^(rd), Hap presented assub-unit antigens, isolated or in combination, are of interest for thedevelopment of a menB vaccine.

TABLE 2 Protective activity in the mouse model of recombinantouter-membrane vesicles. The table summarizes the results obtainedduring one experiment (PV00N050). Sub-unit antigens Survival rate (onday 5) Active mouse protection model PV00N050 (in OF1 mice) Rec sub-unitChallenge strain (+dose) Immuno Antigens CU-385 Specific Abs (fromH44/76 (B:4:P1.19,15) by Elisa Ag sequence) 1.4 10e7 Mean-(GMT) FrpCCa2++ treated 3/5 31477-(27068) FHAB 2/3 refolded 2/5 98200-(73220) FHAB2/3 non 3/5 55939-(35347) refolded Hap N-ter 4/5  9960-(12811) recTbpAon SBAS4 3/5 875-(520) SBAS4 1/5 /

Example 14 Method to Show Synergetic Effect of Vaccine AntigensCombinations

Different recombinant OMVs available (OMVs porA (+) rmp-LbpB, OMVs porA(−) TbpA (+) Hsf (+), OMVs porA (−) TbpA (+), OMVs porA (−) NspA (+),OMVs porA (−) Hsf (+), OMVs porA (−) TbpA (+) NspA (+)) can be testedalone or in combination to determine statistically the bestcombinations, in terms of detecting a synergetic effect of suchcombinations of vaccine candidates. This work can also be performed withcombinations of subunit antigens, as well as combination of subunitantigens+recombinant OMV's. 32 groups of 50F1 mice/group can be injectedand tested for serum bactericidal & opsonic activity, active and passiveprotection in the mouse model (if needbe using suboptimal amounts ofindividual antigens). An indication of synergistic antigen combinationsis if the level of protective conferred after combined immunization ishigher that the sum of individual antigens.

Example 15 Analysis of Hsf and TbpA Content of Outer Membrane VesiclesCoommassie Blue Stained SDS-PAGE

15 μg of protein in outer membrane vesicle preparations withup-regulation of Hsf or TbpA or both Hsf and TbpA, were diluted in asample buffer containing β-mercaptoethanol and heated at 95° C. for 10minutes. The samples were then run on SDS-PAGE polyacrylamide gel (Novex4-20% Tris-glycine 1.5 mm 2Dwell SDS Page), stained in Coomassie bluefor one hour and destained in several washes of destain. Results areshown in FIG. 9, which shows that the level of Hsf and TbpA areconsiderably higher in outer membrane vesicle preparations, derived fromN meningitidis where their level of expression had been enhanced.

Example 16 Immunogenicity of OMVs with Upregulation of Hsf and/or TbpA

Groups of 20 mice were immunised three times with OMV by theintra-muscular route on days 0, 21 and 28. Each innoculation was made upof 5 μg (protein content) of OMVs formulated on AlPO₄ with MPL. The OMVswere derived from N. meningitidis strain H44/76, engineered so thatcapsular polysaccharides and PorA were down regulated. A comparison wasmade of OMVs in which Hsf, TbpA, both Hsf and TbpA or neither wereupregulated. On day 41, blood samples were taken for analysis by ELISAor by serum bactericidal assay.

ELISA to Detect Antibodies Against Hsf

96 well microplates (Nunc, Maxisorb) were coated overnight at 4° C. with100 μl of 1 μg/ml of specific antigen in PBS. After washing with NaC1150 mM Tween 20 0.05%, plates were saturated with 100 μl of PBS-BSA 1%under shaking at room temperature for 30 minutes. Between each step(performed under shaking at room temperature during 30 min and withPBS-BSA 0.2% as diluant buffer), reagents in excess were removed bywashing with NaCl-Tween 20. One hundred micro-liters of diluted serumsamples were added per micro-well. Bound antibodies were recognized by abiotinylated anti-mouse Ig (Prosan) ( 1/2000). The antigen-antibodycomplex was revealed by incubation with streptavidin-biotinylatedperoxidase conjugate (Amersham) ( 1/4000). OrthoPhenileneDiamine/H₂O₂ (4mg/10 ml citrate buffer 0.1M pH 4.5+5 μl H₂O₂) is used to reveal theassay. Plates were incubated for 15 min at room temperature in the darkbefore stoping the reaction by addition of 50 μl of 1N HCl. Theabsorbance was read at 490 nm.

Titre Mid-Point (on pooled sera) g1, blebs TbpA-HSF, IM 15471 g2, blebsTbpA, IM 15.41 g3, blebs HSF, IM 14508 g4, blebs CPS(−)PorA(−), IM — g5,MPL/AlPO4, IM —

The results shown in the table above, show that high and equivalentantibody titres against Hsf were raised by immunisation with OMVs withupregulation of Hsf or both Hsf and TbpA. Virtually no antibody againstHsf could be detected in sera raised after inoculation with adjuvantalone or OMV in which neither Hsf nor TbpA had been upregulated or OMVin which only TbpA had been upregulated.

Example 17 Serum Bactericidal Activity of Antisera Raised Against OMVswith Up-Regulation of Hsf and/or TbpA

The serum bactericidal activity of antisera from the mice inoculatedwith OMVs with upregulation of Hsf, TbpA, both Hsf and TbpA or withoutupregulation were compared in assays using either the homologous strainH44/76 or the heterologous strain Cu385. The serum bactericidal assayhas been shown to show good correlation with the protection and istherefore a good indication of how effective a candidate compositionwill be in eliciting a protective immune response.

Neisseria meningitidis serogroup B wild type strains (H44/76 strain=B:15P1.7, 16 L3, 7, 9 and CU385 strain=B: 4 P1.19, 15 L3, 7, 9) werecultured overnight on MH+1% Polyvitex+1% horse serum Petri dishes at 37°C.+5% CO₂. They were sub-cultured for 3 hours in a liquid TSB mediumsupplemented with 50 μM of Desferal (Iron chelator) at 37° C. undershaking to reach an optical density of approximately 0.5 at 470 nm.

Pooled or individual serum were inactivated for 40 min at 56° C. Serumsamples were diluted 1/100 in HBSS-BSA 0.3% and then serially dilutedtwo fold (8 dilutions) in a volume of 50 nl in round bottom microplates.

Bacteria, at the appropriate OD, were diluted in HBSS-BSA 0.3% to yield1.3 10e4 CFU per ml. 37.5 μl of this dilution was added to the serumdilutions and microplates were incubated for 15 minutes at 37° C. undershaking. Then, 12.5 μl of rabbit complement were added to each well.After 1 hour of incubation at 37° C. and under shaking, the microplateswere placed on ice to stop the killing.

Using the tilt method, 20 μl of each well were platted on MH+1%Polyvitex+1% horse serum Petri dishes and incubated overnight at 37°C.+CO₂. The CFU's were counted and the percent of killing calculated.The serum bactericidal titer is the last dilution yielding ≧50% killing.

H44/76 CU385 OMV GMT % responders GMT % responders CPS (−) PorA (−) 9330% 58 5% CPS (−) PorA (−) 158 40% 108 20% Hsf CPS (−) PorA (−) 327 60%147 30% TbpA CPS (−) PorA (−) 3355 100% 1174 80% Hsf-TbpA

Similar results to those shown in the above table were obtained in twoother similar experiments.

A dramatic increase in the bactericidal titres (GMT) against thehomologous strain and a heterologous strain were seen after vaccinationwith OMV in which both Hsf and TbpA were upregulated. By comparison,bactericidal GMTs measured on mice vaccinated with Hsf or TbpAupregulated OMVs were similar to those obtained with mice vaccinatedwith control OMVs.

The benefit of double up-regulation was also clearly observed in thepercentage of mice producing a significant level of bactericidalantibodies (titres greater than 1/100), particularly in experimentsusing the heterologous strain.

Example 18 Effect of Mixing Anti-Hsf and Anti-TbpA Sera on BactericidalActivity

Groups of 20 mice were immunised three times with OMV by theintra-muscular route on days 0, 21 and 28. Each inoculation was made upof 5 μg (protein content) of OMVs formulated on AlPO4 with MPL. The OMVswere derived from N. meningitidis strain H44/76, engineered so thatcapsular polysaccharides and PorA were down regulated. One group of micewas immunised with control OMVs in which there was no up-regulation ofproteins. In a second group, Hsf expression was up-regulated, in a thirdgroup TbpA expression was up-regulated and in a fourth group, theexpression of both Hsf and TbpA was up-regulated.

The sera were pooled, either using sera from mice in the same group orby mixing sera isolated from the group in with Hsf alone or TbpA alonehad been up-regulated. Serum bactericidal activity was measured for eachof the pooled sera and the results are shown in the table below.

SBA done on pooled sera SBA from mice immunized with titer TbpA-Hsfblebs 774 TbpA blebs 200 Hsf blebs 50 CPS(−) PorA(−) blebs 50 Mixanti-TbpA + anti-Hsf sera 1162

The results in the above table show that mixing of anti-Hsf andanti-TbpA antisera resulted in a much higher serum bactericidal activitythan was achieved by either antisera individually. The synergisticeffect seems to be achieved by the presence of antibodies against bothHsf and TbpA.

Example 19 Truncated Hsf Proteins May Combine Synergistically with TbpA

A series of truncated Hsf constructs were made using standard molecularbiology procedures. These include a construct that encodes amino acids 1to 54 which contains the signal sequence of Hsf and amino acids 134 to592 of Hsf (Tr1Hsf). A second truncated Hsf contained amino acids 1-53of the signal sequence of Hsf followed by amino acids 238-592 of Hsf(Tr2Hsf). These two truncated Hsf constructs and full length Hsf wereintroduced into N Meningitidis B strain MC58 siaD−, Opc−, PorA− so thattheir expression would be up-regulated and outer membrane vesicles wereproduced using the methods described above.

The outer membrane vesicle preparations were adsorbed onto Al(OH)₃ andinjected into mice on days 0, 21 and 28. On day 42, the mice were bledand sera prepared. The sera were mixed with sera from mice vaccinatedwith up-regulated TbpA OMVs and serum bactericidal assays were performedas described above.

Results

Serum Bactericidal titres Group H44/76 CU385 MC58 PorA+ siaD+ 2560025600 MC58 PorA− siaD− Hsf 1530 800 MC58 PorA− siaD− Tr1Hsf 1015 1360MC58 PorA− siaD− Tr2Hsf 50 50 Negative control 50 50 TbpA + MC58 PorA+siaD+ 25600 24182 TbpA + MC58 PorA− siaD− Hsf 2595 1438 TbpA + MC58PorA− siaD− Tr1Hsf 4383 2891 TbpA + MC58 PorA− siaD− Tr2Hsf 1568 742TbpA + Negative control 778 532

The results shown in the above table reveal that the first truncation(Tr1 Hsf) elicits an immune response which is capable of combining withantisera against TbpA to produce a larger serum bactericidal activitythan when full length Hsf is used. However, the extent of the truncationis important and the truncation produced in Tr2 has a deleterious effectcompared to the full length Hsf. The enhanced bactericidal activity ofTr1Hsf was seen against both the strains used.

Example 20 Serum Bactericidal Activity of Antibodies Against TbpA, Hsfand a Third Meningococcal Protein

N. meningitidis strain H66/76 in which PorA and capsular polysaccharideswere down regulated as described above, was used as the backgroundstrain for up-regulating TbpA and Hsf, LbpB, D15, PilQ or NspA using theprocedure described above. Outer membrane vesicles were prepared fromeach strain as described above. Recombinant FHAb, FrpC, FrpA/C and Hapwere made using techniques hereinbefore described and known in the art(as described in PCT/EP99/02766, WO92/01460 and WO98/02547).

The outer membrane vesicle preparations and recombinant proteins wereadsorbed onto Al(OH)₃ and injected into mice on days 0, 21 and 28. Onday 42, the mice were bled and sera prepared. The sera against TbpA andHsf up-regulated OMVs were mixed with sera from mice vaccinated withOMVs containing up-regulated LbpB, D15, PilQ or NspA OMVs or recombinantFHAb, FrpC, FrpA/C or Hap and serum bactericidal assays were performedas described above.

Results

Results are shown in the table below. In assays using the homologousH44/76 stain, the addition of antibodies against a third meningococcalantigen, with the exception of FrpC, did not produce a serumbactericidal titre higher than that produced using antibodies againstTbpA and Hsf alone.

However, the addition of antibodies against a third antigen wasadvantageous in serum bactericidal assays using a heterologous strain.Antibodies against D15 (OMP85), Hap, FrpA/C and LbpB were particularlyeffective at increasing the serum bactericidal titre against the CU385strain.

Serum Bactericidal Titre Antisera Mix H44/76 CU385 anti-TbpA-Hsf andnonimmune sera 5378 2141 anti-TbpA-Hsf and anti-FHA 5260 2563anti-TbpA-Hsf and anti-Hap 4577 5150 anti-TbpA-Hsf and anti-FrpA/C 50344358 anti-TbpA-Hsf and anti-LbpB 5400 4834 anti-TbpA-Hsf and anti-D154823 4657 anti-TbpA-Hsf and anti-PilQ 4708 2242 anti-TbpA-Hsf andanti-NspA 4738 2518 anti-TbpA-Hsf and anti-FrpC 6082 2300

Example 21 Effect of FrpB KO in Outer Membrane Vesicles on their Abilityto Elicit a Bactericidal Immune Response in Homologous and HeterologousStrains

Two strains of H44/76 N. meningitidis were used to prepare outermembrane vesicle preparations as described in WO01/09350, using a 0.1%DOC extraction so that the LOS content was around 20%. Strain B1733 issiaD(−), PorA(−), has upregulation of Tr1 Hsf (example 19) and lgtB isknocked out. Strain B1820 B1733 is siaD(−), PorA(−), has upregulation ofTr1 Hsf, lgtB is knocked out and FrpB is also knocked out. Both strainswere cultured in media supplemented with 60 μM Desferal so that ironregulated proteins such as LbpA/B and TbpA/B are upregulated.

The bleb preparations were adsorbed onto Al(OH)₃ and 5 μg were injectedintramuscularly into groups of 30 mice on day 0 and day 21. Bloodsamples were taken on day 28.

Serum bactericidal assays were carried out on three L3 strains (thehomologous wild type strain H44/76 and two heterologous L3 strains;NZ124 and M97250687), as described in example 17.

Results

Blebs used for inocu- H44/76 M97250687 NZ124 lation GMT SC GMT SC GMT SCB1733 1518 30/30 151 11/30 70  4/29 B1820 781 19/30 1316 24/30 276 19/30GMT indicates the geometric mean titre of the sera in the SBA. SCindicates the number of mice seroconverting (SBA titre >1/100).

The results clearly show that FrpB KO (B 1820) blebs induce a betterheterologous cross-bactericidal response than FrpB(+) blebs (B1733). TheSBA titres were higher and a higher proportion of mice seroconverted instrains M97250687 and NZ124. The results in the homologous strain wasnot quite as good when FrpB was deleted. These data suggest that FrpBdrives the immune response, but since this outer membrane protein ishighly variable, antibodies against this protein are only able to inducekilling of the homologous strain.

1.-12. (canceled)
 13. An immunogenic composition comprising at least oneNeisserial autotransporter antigen, at least one Neisserial Feacquisition protein antigen, and at least one Neisserial outer membraneprotein antigen wherein each of the at least one Neisserialautotransporter antigen, the at least one Neisserial Fe acquisitionprotein antigen, and the at least one Neisseral outer membrane proteinantigen is isolated and wherein said immunogenic composition comprisesboth a subunit composition and an outer membrane vesicle compositionwherein the outer membrane vesicle composition comprises a concentrationof LPS immunotype L3 that is achievable using 0.02-0.4% deoxychoateextraction.
 14. The immunogenic composition of claim 13 wherein all ofthe Neisserial protein antigens are derived from N. meningitidis. 15.The immunogenic composition of claim 13 further comprising one or morebacterial capsular polysaccharides or oligosaccharides.
 16. Theimmunogenic composition of claim 15 wherein the capsular polysaccharidesor oligosaccharides are derived from bacteria selected from the groupconsisting of: Neisseria meningitidis serogroup A, C, Y and W-135. 17.The immunogenic composition of claim 13 further comprising an adjuvant.18. The immunogenic composition of claim 17, wherein the adjuvantcomprises aluminum salts.
 19. The immunogenic composition of claim 18,wherein said aluminum salt is aluminum hydroxide gel.
 20. Theimmunogenic composition of claim 13, wherein the outer membrane proteinantigen is GNA1870.
 21. The immunogenic composition of claim 13, whereinthe Neisseral autotransporter antigen is NadA.
 22. The immunogeniccomposition of claim 13, wherein the Neisseral Fe acquisition proteinantigen is transferrin binding protein related protein.